U.S. patent application number 15/810419 was filed with the patent office on 2018-05-31 for toxoid peptides derived from phenol soluble modulin, delta toxin, superantigens, and fusions thereof.
The applicant listed for this patent is INTEGRATED BIOTHERAPEUTICS, INC.. Invention is credited to Rajan Prasad Adhikari, Mohammad Javad Aman, Frederick Wayne Holtsberg, Hatice Karauzum, Sergey Shulenin.
Application Number | 20180148482 15/810419 |
Document ID | / |
Family ID | 52105236 |
Filed Date | 2018-05-31 |
United States Patent
Application |
20180148482 |
Kind Code |
A1 |
Aman; Mohammad Javad ; et
al. |
May 31, 2018 |
Toxoid Peptides Derived From Phenol Soluble Modulin, Delta Toxin,
Superantigens, and Fusions Thereof
Abstract
The present disclosure provides immunogenic compositions useful
in prevention and treatment of Staphylococcus aureus infection. In
particular, the disclosure provides delta toxin and phenol-soluble
modulin peptides as well as mutants, fragments, variants or
derivatives thereof. The disclosure further provides multivalent
oligopeptides, fusion proteins comprising two or more
staphylococcal proteins, e.g., DT, PSM, alpha hemolysin,
leukocidin, superantigen, or any fragments, variants, derivatives,
or mutants thereof fused together as a single polypeptide in any
order.
Inventors: |
Aman; Mohammad Javad;
(Rockville, MD) ; Adhikari; Rajan Prasad;
(Gaithersburg, MD) ; Shulenin; Sergey; (Point of
Rocks, MD) ; Holtsberg; Frederick Wayne; (Taneytown,
MD) ; Karauzum; Hatice; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INTEGRATED BIOTHERAPEUTICS, INC. |
ROCKVILLE |
MD |
US |
|
|
Family ID: |
52105236 |
Appl. No.: |
15/810419 |
Filed: |
November 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14899993 |
Dec 18, 2015 |
9815872 |
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PCT/US2014/042999 |
Jun 18, 2014 |
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15810419 |
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61836959 |
Jun 19, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 11/00 20180101; A61K 38/00 20130101; C07K 14/31 20130101; A61K
39/085 20130101; A61P 43/00 20180101; A61K 2039/70 20130101; A61P
37/04 20180101; A61K 2039/55572 20130101 |
International
Class: |
C07K 14/31 20060101
C07K014/31; A61K 39/085 20060101 A61K039/085 |
Claims
1-95. (canceled)
96. A multivalent oligopeptide, comprising a fusion of two or more
Staphylococcus aureus-derived superantigen (SAg) polypeptides, or
mutants, fragments, variants, or derivatives thereof arranged in
any order, wherein the two or more Staphylococcus aureus-derived
SAg polypeptides, or mutants, fragments, variants, or derivatives
can be the same or different, and wherein at least one of the two
or more SAg polypeptides, or mutants, fragments, variants, or
derivatives thereof comprises SEQ ID NO: 51
(TSST-1.sub.L30R/D27A/I46A).
97. The oligopeptide of claim 96, wherein two of the two or more
SAg polypeptides, or mutants, fragments, variants, or derivatives
thereof comprise SEQ ID NO: 49 (SEB.sub.L45R/Y89A/Y94A) and SEQ ID
NO: 51 (TSST-1.sub.L30R/D27A/I46A).
98. The oligopeptide of claim 97, wherein the two or more SAg
polypeptides, or mutants, fragments, variants, or derivatives
further comprise SEQ ID NO: 50 (SEA.sub.L48R/D70R/Y92A).
99. The oligopeptide of claim 96, wherein the two or more
Staphylococcus aureus-derived peptides, or mutants, fragments,
variants, or derivatives thereof are associated via a linker.
100-101. (canceled)
102. The oligopeptide of claim 96, further comprising a
heterologous peptide or polypeptide.
103-104. (canceled)
105. The oligopeptide of claim 96, further comprising an
immunogenic carbohydrate.
106-109. (canceled)
110. An isolated polynucleotide comprising a nucleic acid that
encodes the multivalent oligopeptide of claim 96.
111. The polynucleotide of claim 110, further comprising a
heterologous nucleic acid.
112. (canceled)
113. A vector comprising the polynucleotide of claim 110.
114. (canceled)
115. A host cell comprising the vector of claim 113.
116-117. (canceled)
118. A method of producing a multivalent oligopeptide comprising
culturing the host cell of claim 115 and recovering the
oligopeptide.
119. A composition comprising the oligopeptide of claim 96, and a
carrier.
120. The composition of claim 119, further comprising an
adjuvant.
121. (canceled)
122. The composition of claim 119, further comprising an
immunogen.
123-124. (canceled)
125. A method of inducing a host immune response against
Staphylococcus aureus, comprising administering to a subject in
need of the immune response an effective amount of the composition
of claim 119.
126-127. (canceled)
128. A method of preventing or treating a Staphylococcal disease or
infection in a subject comprising administering to a subject in
need thereof the composition of claim 119.
129. The method of claim 128, wherein the infection is a localized
or systemic infection of skin, soft tissue, blood, or an organ, or
is auto-immune in nature.
130. The method of claim 128, wherein the disease is a respiratory
disease.
131. (canceled)
132. The method of claim 128, wherein the disease is sepsis.
133-134. (canceled)
135. The method of claim 128, wherein the subject is a mammal.
136. The method of claim 135, wherein the mammal is a human.
137. (canceled)
138. The method of claim 125, wherein the composition is
administered via intramuscular injection, intradermal injection,
intraperitoneal injection, subcutaneous injection, intravenous
injection, oral administration, mucosal administration, intranasal
administration, or pulmonary administration.
139. A method of producing a vaccine against S. aureus infection
comprising: isolating the multivalent oligopeptide of claim 96; and
combining the peptide, oligopeptide, or any combination thereof
with an adjuvant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation Application of U.S.
National Phase application Ser. No. 14/899,993, filed Dec. 18,
2015, which is a 35 U.S.C. .sctn. 371 US National Stage Application
of International Patent Application PCT/US2014/042999, filed Jun.
18, 2014, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/836,959, filed Jun. 19, 2013, the
disclosures of each of which are incorporated herein by reference
in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence listing
in ASCII text file (Name:
57783_151830_Substitute_SEQ_LIST_ST25.txt; Size: 90,309 bytes (as
measured in MS-Windows.RTM.); and Date of Creation: Nov. 9, 2017)
filed with the application is incorporated herein by reference in
its entirety.
BACKGROUND
[0003] Staphylococcus aureus (SA) is a Gram-positive human pathogen
that causes a wide range of infections from skin and soft tissue
infections (SSTI) to life threatening sepsis and pneumonia. It is a
leading cause of hospital- and community-associated infections
worldwide (Barrio, et al., 2006, Microbes Infect, 8 (8):2068-2074;
Brown, et al., 2009 Clin Microbiol Infect, 15 (2): 156-164; Colin,
et al. 1994, Infect Immun, 62 (8):3184-3188). The range of
pathologies reflects the diverse abilities of SA to escape the
immune response using a plethora of virulence factors: the
superantigenic and pore-forming toxins, coagulase, capsular
polysaccharide, adhesins, proteases, complement inactivating
exoproteins, and other innate response modifiers (Barrio, et al.,
2006, Microbes Infect, 8 (8):2068-2074; Deurenberg and Stobberingh,
2008, Infect Genet Evol, 8 (6):747-763).
[0004] Since its first emergence in the 1960s methicillin-resistant
SA (MRSA) has become endemic in healthcare settings worldwide
(Diep, et al., 2006, J Infect Dis, 193 (11):1495-1503). Since the
1990s, community associated MRSA strains (CA-MRSA) emerged, and are
posing a major global challenge (Bassetti, et al., 2009, Int J
Antimicrob Agents, 34 Suppl 1:S15-19; Bradley, 2005, Semin Respir
Crit Care Med, 26 (6):643-649; Chambers, 2005, N. Engl J Med, 352
(14):1485-1487.). Alpha hemolysin (.alpha.-toxin, Hla) is a major
virulence factor in SA pneumonia and SSTI (Bubeck Wardenburg and
Schneewind, 2008, J Exp Med, 205 (2):287-294; Kennedy, et al.,
2010, J Infect Dis, 202 (7):1050-1058). Recently, cytolytic short
peptides known as phenol soluble modulins (PSMs) were identified as
key virulence factors that lyse neutrophils, the main line of
defense against S. aureus (Wang, et al., 2007, Nat Med, 13
(12):1510-1514). Another related cytolytic short peptide of
staphylococci is known as delta hemolysin or delta toxin
(.delta.toxin) the key marker of S. aureus quorum sensing system
(agr) (Novick, et al., 1993, EMBO J, 12 (10):3967-3975). A recent
epidemiological study in a cohort of patients with SA bacteremia
shows inverse correlation between probability of sepsis and
pre-existing antibodies to Hla, PSM-.alpha.3, as well as
.delta.-toxin (Adhikari, et al., 2012, J Infect Dis, 206
(6):915-923).
[0005] Staphylococcal superantigens (SAgs) induce a massive release
of cytokines and chemokines, enhanced expression and activation of
adhesion molecules, increased T-cell proliferation, and ultimately
T-cell apoptosis/anergy. This sequence of events can culminate in
Toxic Shock Syndrome (TSS), a life threatening condition (Todd, et
al., 1978, Lancet, 2 (8100):1116-1118) characterized by rash,
hypotension, fever, and multisystem dysfunction (Bohach, et al.,
1990, Crit Rev Microbiol, 17 (4):251-272.). A major challenge in
development of multivalent S. aureus vaccines including
superantigens is that there are more than 20 different SAgs and
there is a wide range of variability in SAg presence in clinical
isolates because most SEs are on mobile genetic elements, such as
plasmids or pathogenicity islands (Staphylococcal enterotoxin K
(sek), (Staphylococcal enterotoxin Q seq), lysogenic phages
(Staphylococcal enterotoxin A (sea), or antibiotic resistance
cassettes, like SCCmec (Staphylococcal enterotoxin H (seh) (Omoe,
et al., 2002, J Clin Microbiol, 40 (3):857-862). Based on an
extensive literature review encompassing over 6000 clinical
isolates, the most widely represented Superantigens (Sags) appear
to be toxic shock syndrome toxin 1 (TSST-1) and (Staphylococcal
enterotoxin C (SEC), followed by (Staphylococcal enterotoxin A
(SEA), (Staphylococcal enterotoxin D (SED), and (Staphylococcal
enterotoxin B (SEB). More recent studies show the emergence of
(Staphylococcal enterotoxin K (SEK) and (Staphylococcal
enterotoxinQ (SEQ), primarily due to circulation of the USA300
clone. Attenuated Superantigen toxoids for (Staphylococcal
enterotoxin (SEA), (Staphylococcal enterotoxin B (SEB), and TSST-1
have been designed and tested in animal models of toxic shock.
These mutants are deficient in binding to MHC class II protein and
therefore lack superantigenic activity (subject of U.S. Pat. Nos.
6,713,284; 6,399,332; 7,087,235; 7,750,132, 7,378,257, and
8,067,202). A simplified superantigen toxoid vaccine capable of
inducing broad neutralizing antibodies is therefore highly is
needed to be practical for inclusion into a multivalent S. aureus
vaccine.
[0006] Phenol Soluble Modulins (PSMs) and
Delta-Hemolysin(.delta.-Toxin):
[0007] S. aureus secretes four short (.about.20 amino acids,
.alpha.-type) and two longer (.about.40 amino acids, .beta.-type)
cytolytic peptides, known as phenol soluble modulin (PSM) (Wang, et
al., 2007, Nat Med, 13 (12):1510-1514). In addition, SA produces
.delta.-toxin, which is similar to the .alpha.-type PSMs. These
genes are expressed in all S. aureus strains under the control of
the agr system (Wang, et al., 2007, Nat Med, 13 (12):1510-1514).
Recently, a novel PSM (PSM-mec) has been also identified within the
staphylococcal methicillin resistance mobile genetic element SCCmec
(Chatterjee, et al., 2011, PLoS One, 6 (12):e28781; Kaito, et al.,
2011, PLoS Pathog, 7 (2):e1001267) suggesting that horizontal
transfer of these toxins can contribute to MRSA virulence. PSMs are
lytic towards neutrophils, the first line of host defense against
SA (Wang, et al., 2007, Nat Med, 13 (12):1510-1514). Furthermore,
recent studies indicate a synergistic effect on hemolytic activity
of .beta.-hemolysin (Cheung, et al., 2012, Microbes Infect, 14
(4):380-386). A key role of PSMs in pathogenesis has been shown in
mouse models of bacteremia and SSTI using deletion mutants (Wang,
et al., 2007, Nat Med, 13 (12):1510-1514). Furthermore, a recent
report suggests a key role for PSMs in biofilm formation
(Periasamy, et al., 2012, Proc Natl Acad Sci USA, 109
(4):1281-1286). Among the PSMs, PSM-.alpha.3 plays the most
prominent role in S. aureus pathogenesis (Wang, et al., 2007, Nat
Med, 13 (12):1510-1514).
[0008] .delta.-Toxin is encoded by the hld gene located within
RNAIII transcript of agr locus. RNAIII is a regulatory RNA and
plays a major role in regulation of SA quorum-sensing system for
the expression of various virulence genes (Novick, 2003, Mol
Microbiol, 48 (6):1429-1449; Novick, et al., 1993, EMBO J, 12
(10):3967-3975). With increased expression of RNAIII, the level of
extracellular .delta.-toxin is increased reaching almost half the
amount of total exoproteins at the stationary phase (Kreger, et
al., 1971, Infect Immun, 3 (3):449-465). The hld.sup.-/- mutant of
the CA-MRSA strain MW2 exhibited attenuated virulence in mouse
bacteremia model (Wang, et al., 2007, Nat Med, 13 (12):1510-1514).
A recent study also revealed that .delta.-toxin increases the cell
number/unit area of colony by inhibiting colony spreading,
resulting in a thicker giant colony and promotes the
compartmentalization of SA colonies leading to efficient
colonization (Omae, et al., 2012, J Biol Chem, 287
(19):15570-15579). Thus .delta.-toxin also appears to modulate the
physical state of SA colonies. .delta.-toxin also plays an
important role in the escape of S. aureus from phago-endosomes of
human epithelial and endothelial cells in the presence of
beta-toxin (Giese, et al., 2011, Cell Microbiol, 13 (2):316-329) by
acting as a costimulator of human neutrophil oxidative burst
(Schmitz, et al., 1997, J Infect Dis, 176 (6): 1531-1537).
[0009] S. aureus Alpha Hemolysin (Hla):
[0010] The pore forming toxins form oligomeric beta barrel pores in
the plasma membrane and play an important role for bacterial spread
and survival, immune evasion and tissue destruction. SA alpha-toxin
(Hla) (Bhakdi and Tranum-Jensen, 1991, Microbiol Rev, 55
(4):733-751) targets many cells such as lymphocytes, macrophages,
pulmonary epithelial cells and endothelium, and erythrocytes
(Bhakdi and Tranum-Jensen, 1991, Microbiol Rev, 55 (4):733-751;
McElroy, et al., 1999, Infect Immun, 67 (10):5541-5544). Several
lines of evidence validate Hla as a prime vaccine target for
prevention of S. aureus infection or complications: i) hla is
encoded by a chromosomal determinant (Brown and Pattee, 1980,
Infect Immun, 30 (1):36-42), and expressed in most SA isolates
(Aarestrup, et al., 1999, APMIS, 107 (4):425-430; Bhakdi and
Tranum-Jensen, 1991, Microbiol Rev, 55 (4):733-751; Husmann, et
al., 2009, FEBS Lett, 583 (2):337-344; Shukla, et al., 2010, J Clin
Microbiol, 48 (10):3582-3592); ii) A partially attenuated Hla
(H35L) and antibodies to Hla protect mice against SA pneumonia and
skin infections (Kennedy, et al., 2010, J Infect Dis, 202
(7):1050-1058; Bubeck Wardenburg and Schneewind, 2008, J Exp Med,
205 (2):287-294; Ragle and Bubeck Wardenburg, 2009, J Infect Dis,
176 (6):1531-1537); iii) Antibodies to H35L protect mice from toxin
and partially protect against bacterial challenge (Menzies and
Kernodle, 1996; Infect Immun, 64 (5):1839-1841). While the H35
mutation largely abrogates the lytic activity of Hla, a single
point mutation is not considered sufficiently safe to be developed
as vaccine for human use.
[0011] WO 2012/109167A1 describes a rationally designed mutant
vaccine for Hla referred to as AT62. Immunization of mice with AT62
protected the animals against S. aureus lethal sepsis and pneumonia
(Adhikari, et al., 2012, PLoS One, 7 (6):e38567). Furthermore,
antibodies raised against AT62 protected mice from lethal sepsis
induced by S. aureus (Adhikari, et al., 2012, PLoS One, 7
(6):e38567).
[0012] Panton-Valentine Leukocidin (PVL):
[0013] PVL is a member of a family of bicomponent cytolytic toxins
known as leukotoxins that is produced by several CA-MRSA lineages
(Diep and Otto, 2008, Trends Microbiol, 16 (8):361-369). The
bi-component hemolysins and leukotoxins, play an important role in
staphylococcal immune evasion. These toxins kill key immune cells
and cause tissue destruction, thereby often weakening the host
during the first stage of infection and promoting bacterial
dissemination and metastatic growth in distant organs. The two PVL
components LukS-PV and LukF-PV are secreted separately, and form
the pore-forming octameric complex upon binding of LukS-PV to its
receptor and subsequent binding of LukF-PV to LukS-PV (Miles, et
al., 2002, Protein Sci, 11 (4):894-902; Pedelacq, et al., 2000,
Proc Natl Acad Sci USA, 109 (4):1281-1286). Targets of PVL are
polymorphonuclear phagocytes (PMN), monocytes, and macrophages.
Epidemiologically, PVL is associated with primary skin infections,
such as furunculosis and severe necrotizing pneumonia that rapidly
progresses towards acute respiratory distress syndrome. The role of
PVL in skin, bone, and lung infections has been shown in animal
models (Brown, et al., 2009 Clin Microbiol Infect, 15 (2):156-164;
Cremieux, et al., 2009, PLoS ONE, 4 (9):e7204; Diep, et al., 2010,
Proc Natl Acad Sci USA, 107 (12):5587-5592; Tseng, et al., 2009
PLoS ONE, 4 (7):e6387; Varshney, et al., 2010, J Infect Dis 1;
201(1):92-6). PVL-positive CA-MRSA affect healthy children or young
adults that had neither any recent contact with health care
facilities nor with any risk factors with a mortality of up to 75%
(Gillet, et al., 2002, Lancet, 359 (9308):753-759; Lina, et al.,
1999, Clin Infect Dis, 29 (5):1128-1132).
[0014] PCT application No. PCT/US12/67483 discloses rationally
designed mutants vaccine for LukS-PV and LukF-PV. Immunization of
mice with these mutants protected the animals against S. aureus
lethal sepsis (Karauzum, et al., 2013, PLoS ONE, 8 (6):e65384).
Furthermore, antibodies raised against LukS-PV mutant protected
mice from lethal sepsis induced by S. aureus (Karauzum, et al.,
2013, PLoS ONE, 8 (6):e65384).
[0015] Staphylococcus aureus Enterotoxins:
[0016] Superantigens (SAgs) constitute a large family of pyrogenic
toxins composed of staphylococcal enterotoxins (SEs) and toxic
shock syndrome toxin 1 (TSST-1) (Johns and Khan, 1988, J Bacteriol,
170 (9):4033-4039). In contrast to conventional antigens that
undergo proteolytic processing by antigen presenting cells and are
presented as MHC/peptide complex to T cells, SAgs cross link TCR
with MHC Class II and activate up to 30% of T cells (Choi, et al.,
1989, Proc Natl Acad Sci USA, 86 (22):8941-8; Marrack and Kappler,
1990, Science, 248 (4959):1) leading to massive release of
cytokines and chemokines, enhanced expression as well as activation
of cell-adhesion molecules, increased T-cell proliferation, and
eventually T-cell apoptosis/anergy. This sequence of events can
culminate in Toxic Shock Syndrome (TSS), a life threatening
condition (Todd, et al., 1978, Lancet, 2 (8100):1116-1118)
characterized by rash, hypotension, fever, and multisystem
dysfunction (Bohach, et al., 1990, Crit Rev Microbiol, 17
(4):251-272). Antibodies play an important role in protection
against TSS (Bonventre, et al., 1984, J Infect Dis, 150
(5):662-666; Notermans, et al., 1983, J Clin Microbiol, 18
(5):1055-1060), thus individuals that do not seroconvert towards
the offending toxin due to hyporesponsive T-cells (Mahlknecht, et
al., 1996, Hum Immunol, 45 (1):42-4) and/or T-cell dependent B-cell
apoptosis (Hofer, et al., 1996, Proc Natl Acad Sci USA, 93
(11):5425-5430) are more likely to experience recurring bouts.
Clonal deletion of CD4 T cells can further impair effective
antibody response to other S. aureus antigens. Furthermore, at
lower non-TSS inducing concentrations SAgs impact the virulence of
S. aureus strains through induction of a local excessive
inflammatory response. Attenuated mutants of SEs and TSST-1 have
been developed that are deficient in binding to MHC-class II
molecules. These mutants can serve as a vaccine for S. aureus
infections as well as toxic shock syndrome by inducing neutralizing
antibodies against superantigens.
SUMMARY
[0017] In one aspect, the disclosure provides a recombinant peptide
that can include a Staphylococcus aureus delta toxin peptide or a
mutant, fragment, variant or derivative thereof (DT); a
Staphylococcus aureus phenol soluble modulin peptide or a mutant,
fragment, variant or derivative thereof (PSM); or a fusion of a DT
and a PSM; where the peptide is attenuated relative to wild-type
DT, PSM, or both, and where the peptide can elicit an
anti-Staphylococcus aureus immune response when administered to a
subject. In certain aspects, surfactant properties of the DT, PSM,
or both, is reduced, while maintaining immunogenicity. In certain
aspects, hydrophobicity is reduced while maintaining the peptide's
alpha-helical structure. In certain aspects, at least one
hydrophobic amino acid is replaced with a less hydrophobic amino
acid, for example, valine (V), leucine (L), isoleucine (I),
phenylalanine (F), or methionine (M) can be replaced with glycine
or alanine.
[0018] The disclosure provides a DT that includes the amino acid
sequence
MAQDX.sub.5X.sub.6STX.sub.9GDX.sub.12X.sub.13KWX.sub.16X.sub.17DTX.sub.20-
NKFTKK (SEQ ID NO: 39), where at least one of X X.sub.5, X.sub.6,
X.sub.9, X.sub.12, X.sub.13, X.sub.16, X.sub.17, or X.sub.20
includes an amino acid substitution relative to SEQ ID NO: 1, and
where X.sub.5 is isoleucine (I), glycine (G) or alanine (A),
X.sub.6 is I, G, or A, X.sub.9 is I, G, or A, X.sub.12 is leucine
(L), G, or V, X.sub.13 is valine (V), G, or A, X.sub.16 is I, G, or
A, X.sub.17 is I, G, or A, and X.sub.20 is V, G, or A. In certain
aspects the peptide includes the amino acid sequence SEQ ID NO: 2.
In certain aspects the peptide includes the amino acid sequence SEQ
ID NO: 4. In certain aspects the peptide includes the amino acid
sequence SEQ ID NO: 3. In certain aspects the peptide includes the
amino acid sequence SEQ ID NO: 5.
[0019] The disclosure further provides a PSM that can be
PSM.alpha.1, PSM.alpha.2, PSM.alpha.3, PSM.alpha.4, PSM.beta.1,
PSM.beta.2, PSM-mec, or any combination of two or more PSMs.
[0020] The disclosure provides a PSM.alpha.1 mutant including the
amino acid sequence
X.sub.1GX.sub.3X.sub.4AGX.sub.7X.sub.8KX.sub.10X.sub.11KSX.sub.14X.sub.15-
EQX.sub.18TGK (SEQ ID NO: 40), where at least one of X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, or X.sub.18 includes an amino acid substitution relative
to SEQ ID NO: 38, and where X.sub.1 is methionine (M), G, or A,
X.sub.3 is I, G, or A, X.sub.4 is I, G, or A, X.sub.7 is I, G, or
A, X.sub.8 is I, G, or A, X.sub.10 is V, G, or A, X.sub.11 is I, G,
or A, X.sub.14 is L, G, or A, X.sub.15 is I, G, or A, and X.sub.18
is F, G, or A. The disclosure provides a PSM.alpha.2 mutant
including the amino acid sequence
X.sub.1GX.sub.3X.sub.4AGX.sub.7X.sub.8KX.sub.10X.sub.11KGX.sub.14X.sub.15-
EKX.sub.18TGK (SEQ ID NO: 41), where at least one of X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, or X.sub.18 includes an amino acid substitution relative
to SEQ ID NO: 12, and where X.sub.1 is M, G, or A, X.sub.3 is I, G,
or A, X.sub.4 is I, G, or A, X.sub.7 is I, G, or A, X.sub.8 is I,
G, or A, X.sub.10 is F, G, or A, X.sub.11 is I, G, or A, X.sub.14
is L, G, or A, X.sub.15 is I, G, or A, and X.sub.18 is F, G, or A.
The disclosure provides a PSM.alpha.3 mutant including the amino
acid sequence
X.sub.1EX.sub.3X.sub.4AKX.sub.7X.sub.8KX.sub.10X.sub.11KDX.sub.14X.sub.15-
GKX.sub.18X.sub.19GNN (SEQ ID NO: 42), where at least one of
X.sub.1, X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11,
X.sub.14, X.sub.15, X.sub.18, or X.sub.19 includes an amino acid
substitution relative to SEQ ID NO: 6, and where X.sub.1 is M, G,
or A, X.sub.3 is F, G, or A, X.sub.4 is V, G, or A, X.sub.7 is L,
G, or A, X.sub.8 is F, G, or A, X.sub.10 is F, G, or A, X.sub.11 is
F, G, or A, X.sub.14 is L, G, or A, X.sub.15 is L, G, or A,
X.sub.18 is F, G, or A, and X.sub.19 is L, G, or A. In certain
aspects the peptide includes the amino acid sequence SEQ ID NO: 7.
In certain aspects the peptide includes the amino acid sequence SEQ
ID NO: 9. In certain aspects the peptide includes the amino acid
sequence SEQ ID NO: 8. In certain aspects the peptide includes the
amino acid sequence SEQ ID NO: 10. In certain aspects the peptide
includes the amino acid sequence e SEQ ID NO: 11. The disclosure
provides a PSM.alpha.4 mutant including the amino acid sequence
X.sub.1AX.sub.3X.sub.4GTX.sub.7X.sub.8KX.sub.10X.sub.11KAX.sub.14X.sub.15-
DX.sub.17X.sub.18AK (SEQ ID NO: 43), where at least one of X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, X.sub.17, or X.sub.18 includes an amino acid substitution
relative to SEQ ID NO: 14, and where X.sub.1 is M, G, or A, X.sub.3
is I, G, or A, X.sub.4 is V, G, or A, X.sub.7 is I, G, or A,
X.sub.8 is I, G, or A, X.sub.10 is I, G, or A, X.sub.11 is I, G, or
A, X.sub.14 is I, G, or A, X.sub.15 is I, G, or A, X.sub.17 is I,
G, or A, and X.sub.18 is F, G, or A. The disclosure provides a
PSM.beta.1 mutant including the amino acid sequence
MEGX.sub.4X.sub.5NAX.sub.8KDTX.sub.12TAAX.sub.16NNDGAKLGTSIVNIVENGVGLLSKL-
FGF (SEQ ID NO: 44), where at least one of X.sub.4, X.sub.5,
X.sub.8, X.sub.12, or X.sub.16 includes an amino acid substitution
relative to SEQ ID NO: 15, and where X.sub.4 is L, G, or A, X.sub.5
is F, G, or A, X.sub.8 is I, G, or A, X.sub.12 is V, G, or A, and
X.sub.16 is I, G, or A. The disclosure provides a PSM.beta.2 mutant
including the amino acid sequence
MTGX.sub.4AEAX.sub.8ANTX.sub.12QAAQQHDSVKX.sub.23GTSIVDIVANGVGLL-
GKLFGF (SEQ ID NO: 45), where at least one of X.sub.4, X.sub.8,
X.sub.12, or X.sub.23 includes an amino acid substitution relative
to SEQ ID NO: 16, and where X.sub.4 is L, G, or A, X.sub.8 is I, G,
or A, X.sub.12 is V, G, or A, and X.sub.23 is L, G, or A.
[0021] The disclosure further provides a multivalent oligopeptide
that includes a fusion of two or more Staphylococcus aureus-derived
peptides, or mutants, fragments, variants, or derivatives thereof
arranged in any order, where the two or more Staphylococcus
aureus-derived peptides, or mutants, fragments, variants, or
derivatives thereof can be the same or different, and where the
multivalent oligopeptide includes two or more of: a wild-type DT,
or a mutant DT, e.g., as described herein; a wild-type PSM, or a
mutant PSM, e.g., as described herein; an alpha hemolysin
polypeptide or mutant, fragment, variant, or derivative thereof; a
leukocidin polypeptide or mutant, fragment, variant, or derivative
thereof; or a superantigen (SAg) polypeptide, or mutant, fragment,
variant, or derivative thereof.
[0022] A multivalent oligopeptide as provided herein can include an
alpha hemolysin polypeptide or mutant, fragment, variant, or
derivative thereof such as the amino acid sequence SEQ ID NO: 46
(AT-62). A multivalent oligopeptide as provided herein can include
a Panton-Valentine leukocidin (PVL) LukS-PV subunit such as an
amino acid sequence at least 90% identical to SEQ ID NO: 47, a
LukS-Mut9 (SEQ ID NO: 54), a Panton-Valentine leukocidin (PVL)
LukF-PV subunit such as an amino acid sequence at least 90%
identical to SEQ ID NO: 48, a LukF-Mut-1 (SEQ ID NO: 55), or a
combination thereof. A multivalent oligopeptide as provided herein
can include a staphylococcal enterotoxin B (SEB) or mutant,
fragment, variant, or derivative thereof such as an amino acid
sequence at least 90% identical to SEQ ID NO: 49, a staphylococcal
enterotoxin A (SEA) or mutant, fragment, variant, or derivative
thereof such as an amino acid sequence at least 90% identical to
SEQ ID NO: 50, a staphylococcal toxic shock syndrome toxin-1 or
mutant, fragment, variant, or derivative thereof such as an amino
acid sequence at least 90% identical to SEQ ID NO: 51, or any
combination thereof.
[0023] A multivalent oligopeptide as provided herein can include
linkers connecting the two or more Staphylococcus aureus-derived
peptides, or mutants, fragments, variants, or derivatives thereof
are associated via a linker. The linker can include, e.g., at least
one, but no more than 50 amino acids selected from the group
consisting of glycine, serine, alanine, and a combination thereof.
In certain aspects the linker includes (GGGS).sub.n (SEQ ID NO: 56)
or (GGGGS).sub.n (SEQ ID NO: 57), where n is a integer from 1 to
10. Exemplary multivalent oligopeptides provided by the disclosure
include, without limitation, the amino acid sequence SEQ ID NO: 18,
SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID
NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO. 34, SEQ ID NO: 35,
SEQ ID NO: 36, SEQ ID NO: 37, or any combination thereof.
[0024] Any peptide or oligopeptide provided by the disclosure can
further include a heterologous peptide or polypeptide including,
but not limited to a His-tag, a ubiquitin tag, a NusA tag, a chitin
binding domain, a B-tag, a HSB-tag, green fluorescent protein
(GFP), a calmodulin binding protein (CBP), a galactose-binding
protein, a maltose binding protein (MBP), cellulose binding domains
(CBD's), an avidin/streptavidin/Strep-tag, trpE, chloramphenicol
acetyltransferase, lacZ (.beta.-Galactosidase), a FLAG.TM. peptide,
an S-tag, a T7-tag, a fragment of any of the heterologous peptides,
or a combination of two or more of the heterologous peptides. In
certain aspects the heterologous peptide or polypeptide includes an
immunogen, a T-cell epitope, a B-cell epitope, a fragment thereof,
or a combination thereof.
[0025] Any peptide or oligopeptide provided by the disclosure can
further include an immunogenic carbohydrate, e.g., a saccharide.
The immunogenic carbohydrate can be a capsular polysaccharide or a
surface polysaccharide. The immunogenic carbohydrate can include,
without limitation, capsular polysaccharide (CP) serotype 5 (CP5),
CP8, poly-N-acetylglucosamine (PNAG), poly-N-succinyl glucosamine
(PNSG), Wall Teichoic Acid (WTA), Lipoteichoic acid (LTA), a
fragment of any of the immunogenic carbohydrates, and a combination
of two or more of the immunogenic carbohydrates. In certain aspects
the immunogenic carbohydrate is conjugated to the oligopeptide.
[0026] The disclosure further provides an isolated polynucleotide
that includes a nucleic acid any peptide or oligopeptide provided
herein, and any combination thereof. In certain aspects the
polynucleotide can further include a heterologous nucleic acid,
e.g., a promoter operably associated with the nucleic acid encoding
the multivalent oligopeptide, DT, PSM, or any combination thereof.
The disclosure provides a vector that includes the polynucleotide
provided by the disclosure, e.g., a plasmid. The disclosure
provides a host cell that includes a vector provided by the
disclosure. The host cell can be, e.g., a bacterium, e.g.,
Escherichia coli, an insect cell, a mammalian cell or a plant
cell.
[0027] The disclosure further provides a method of producing a
multivalent oligopeptide, DT, PSM, or any combination thereof as
provided herein, that includes culturing a host cell and recovering
the oligopeptide, DT, PSM, or any combination thereof.
[0028] The disclosure further provides a composition that includes
any peptide, oligopeptide, or combination thereof provided by the
disclosure, and a carrier. In certain aspects the composition
further includes an adjuvant that can be, without limitation, alum,
aluminum hydroxide, aluminum phosphate, or a glucopyranosyl lipid
A-based adjuvant. In certain aspects the composition further
includes an immunogen that can be, without limitation, a bacterial
antigen such as a pore forming toxin, a superantigen, a cell
surface protein, a fragment of any of the bacterial antigens, or a
combination of two or more of the bacterial antigens.
[0029] The disclosure further provides a method of inducing a host
immune response against Staphylococcus aureus that includes
administering to a subject in need of the immune response an
effective amount of the composition of the disclosure. In certain
aspects the immune response is an antibody response, an innate
response, a humoral response, a cellular response, and a
combination of two or more of the immune responses. The disclosure
further provides a method of preventing or treating a
Staphylococcal, Streptococcal, or Enterococcal disease or infection
in a subject that includes administering to a subject in need
thereof the composition of the disclosure. The infection can be,
e.g., a localized or systemic infection of skin, soft tissue,
blood, or an organ, or can be auto-immune in nature. In certain
aspects the disease is a respiratory disease, e.g., pneumonia. In
certain aspects the disease is sepsis.
[0030] A subject to be treated by the methods of the disclosure can
be a mammal, e.g., a human, bovine, or canine. The composition can
be administered to the subject via intramuscular injection,
intradermal injection, intraperitoneal injection, subcutaneous
injection, intravenous injection, oral administration, mucosal
administration, intranasal administration, or pulmonary
administration.
[0031] The disclosure further provides a method of producing a
vaccine against S. aureus infection that includes: isolating a
peptide or oligopeptide as provided by this disclosure, or any
combination thereof; and combining the peptide, oligopeptide, or
any combination thereof with an adjuvant.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0032] FIG. 1A: Helical wheel representation of PSM-.alpha.1-4,
PSM-.beta.1&2, and .delta.-toxin showing the hydrophobic and
hydrophilic surfaces. VILIFIIIIM (SEQ ID NO: 58); FILIFIIIM (SEQ ID
NO: 59); FFLLFFVLFM (SEQ ID NO: 60); IIIIFIVIIM (SEQ ID NO: 61);
LAIMVF (SEQ ID NO: 62); LAIMVA (SEQ ID NO: 63); and IIIVVII (SEQ ID
NO: 64).
[0033] FIG. 1B: Mutation strategies for the .delta.-toxin and
PSM-.alpha.3. Amino acids L and V, conserved in hydrophobic faces,
are replaced either by ala (A) or Gly (G) individually or in
combination (gly-ala). The predicted decrease in hydrophobicity and
hydrophobic moment are shown for each mutant. Polar amino acids
are: (D, E: - charge) and (R, K: + charge); Other polar amino acids
are (Q, N, T); Hydrophobic amino acids are (F, L, I) and such that
are not disturbing hydrophobicity are (A) or (P) for their aromatic
side-chain.
[0034] FIG. 1C: Helical wheel representation of PSM-mec showing the
hydrophobic and hydrophilic surfaces. MDFTGVITSIIDLIKTCIQAFG (SEQ
ID NO: 65) and LVCIFIIII (SEQ ID NO: 66).
[0035] FIG. 2: Delta toxin mutants toxicity assay. WT and mutants
at concentration of 12.5 .mu.g/ml were tested in different % of
horse RBC. Hemolysis ODs were measured at 416 nm.
[0036] FIG. 3: DT-ala2 mutant is highly attenuated while retaining
binding to human neutralizing antibodies (compare 3.sup.rd and
7.sup.th bar).
[0037] FIG. 4: PSM mutants toxicity assay. WT and mutants at
different concentration were tested in different 5% of horse RBC.
Hemolysis ODs were measured at 416 nm.
[0038] FIG. 5A: Schematic of three fusion proteins generated with
flexible linkers (G4S, denoted as L) and a 6.times.His tag.
[0039] FIG. 5B: Schematic of the secondary structure of AT62-DT-PSM
construct.
[0040] FIG. 5C: Candidate peptides were expressed in E. coli and
tested by Western blot using an AT62 specific mAb. Lanes 1 and 2:
AT62_DT_PSM; 3 and 4: AT62_PSM; 5 and 6: AT62_DT (Two clones for
each construct are shown).
[0041] FIG. 6: Antibody response of mice immunized with AT62-PSM
and AT62-DT against wild type peptides or full length alpha
hemolysin (Hla).
[0042] FIG. 7: Schematic of three fusion proteins: AT-62, rSEB and
DT generated with flexible linkers (G4S, denoted as L).
[0043] FIG. 8: Human antibodies to SEA, SEB, and TSST-1 were
affinity purified from human IVIG and used in toxin neutralization
assays with human PBMC against the SAgs shown on the X axes. The Y
axis shows the molar ratio of antibody to toxin required for 50%
inhibition of superantigenic activity of the respective SAg on the
X axis. The panel titled Cocktail shows the activity of the
combination of the three antibodies. Note that lower molar ratio
indicates higher neutralizing activity towards the respective
toxin.
DETAILED DESCRIPTION
I. Definitions
[0044] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, "a polynucleotide," is
understood to represent one or more polynucleotides. As such, the
terms "a" (or "an"), "one or more," and "at least one" can be used
interchangeably herein.
[0045] Furthermore, "and/or" where used herein is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. Thus, the term and/or" as
used in a phrase such as "A and/or B" herein is intended to include
"A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the
term "and/or" as used in a phrase such as "A, B, and/or C" is
intended to encompass each of the following embodiments: A, B, and
C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A
(alone); B (alone); and C (alone).
[0046] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0047] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, amino acid sequences are written left to right in amino
to carboxy orientation. The headings provided herein are not
limitations of the various aspects or embodiments of the
disclosure, which can be had by reference to the specification as a
whole. Accordingly, the terms defined immediately below are more
fully defined by reference to the specification in its
entirety.
[0048] Wherever embodiments are described with the language
"comprising," otherwise analogous embodiments described in terms of
"consisting of" and/or "consisting essentially of" are also
provided.
[0049] Amino acids are referred to herein by their commonly known
three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter
codes.
[0050] The terms "nucleic acid" or "nucleic acid fragment" refers
to any one or more nucleic acid segments, e.g., DNA or RNA
fragments, present in a polynucleotide or construct. Two or more
nucleic acids of the disclosure can be present in a single
polynucleotide construct, e.g., on a single plasmid, or in separate
(non-identical) polynucleotide constructs, e.g., on separate
plasmids. Furthermore, any nucleic acid or nucleic acid fragment
can encode a single polypeptide, e.g., a single antigen, cytokine,
or regulatory polypeptide, or can encode more than one polypeptide,
e.g., a nucleic acid can encode two or more polypeptides. In
addition, a nucleic acid can encode a regulatory element such as a
promoter or a transcription terminator, or can encode a specialized
element or motif of a polypeptide or protein, such as a secretory
signal peptide or a functional domain.
[0051] The term "polynucleotide" is intended to encompass a
singular nucleic acid or nucleic acid fragment as well as plural
nucleic acids or nucleic acid fragments, and refers to an isolated
molecule or construct, e.g., a virus genome (e.g., a non-infectious
viral genome), messenger RNA (mRNA), plasmid DNA (pDNA), or
derivatives of pDNA (e.g., minicircles as described in (Darquet,
A-M et al., Gene Therapy 4:1341-1349, 1997) comprising a
polynucleotide. A polynucleotide can be provided in linear (e.g.,
mRNA), circular (e.g., plasmid), or branched form as well as
double-stranded or single-stranded forms. A polynucleotide can
comprise a conventional phosphodiester bond or a non-conventional
bond (e.g., an amide bond, such as found in peptide nucleic acids
(PNA)).
[0052] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and comprises any chain or chains of two or more
amino acids. Thus, as used herein, a "peptide," an "oligopeptide,"
a "dipeptide," a "tripeptide," a "protein," an "amino acid chain,"
an "amino acid sequence," "a peptide subunit," or any other term
used to refer to a chain or chains of two or more amino acids, are
included in the definition of a "polypeptide," (even though each of
these terms can have a more specific meaning) and the term
"polypeptide" can be used instead of, or interchangeably with any
of these terms. The term further includes polypeptides which have
undergone post-translational modifications, for example,
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, or modification by non-naturally occurring amino
acids.
[0053] The terms "delta toxin" or "DT" as used herein, and unless
otherwise indicated, encompass wild-type delta toxin peptides as
well as mutants, fragments, variants or derivatives thereof. The
terms "phenol-soluble modulin," "PSM," PSM.alpha.1, PSM.alpha.2,
PSM.alpha.3, PSM.alpha.4, PSM.beta.1, PSM.beta.2, and PSM-mec as
used herein, and unless otherwise indicated, encompass wild-type
phenol-soluble modulin peptides as well as mutants, fragments,
variants or derivatives thereof. By "corresponding wild-type DT" or
"corresponding wild-type PSM" is meant the native DT or PSM peptide
from which a mutant peptide subunit was derived.
[0054] The term "multivalent oligopeptide" as used herein refers to
a fusion protein comprising two or more staphylococcal proteins,
e.g., DT, PSM, alpha hemolysin, leukocidin, superantigen, or any
fragments, variants, derivatives, or mutants thereof fused together
as a single polypeptide in any order. An oligopeptide can include
other heterologous peptides as described elsewhere herein. Other
peptides for inclusion in a multivalent oligopeptide provided
herein include various other staphylococcal toxins or mutants
fragments, variants, or derivatives thereof, described elsewhere
herein or in PCT Publication Nos. WO 2012/109167A1 and WO
2013/082558 A1, which are both incorporated by reference herein in
their entireties.
[0055] This disclosure provides specific DT and PSM peptides as
well as multivalent oligopeptides that can, but do not necessarily
include either a wild-type or mutant DT, PSM, or any combination
thereof. The collection of peptides and oligopeptides provided by
the disclosure are collectively referred to herein as a
"multivalent oligopeptide, DT, and/or PSM," or a "multivalent
oligopeptide, DT, PSM, or any combination thereof." These
collective references are meant to include, without limitation, any
one peptide or oligopeptide as provided herein, or two, three,
four, or more peptides or oligopeptides as provided herein.
[0056] The terms "fragment," "mutant," "derivative," or "variant"
when referring to a multivalent oligopeptide, DT, and/or PSM of the
present disclosure include any polypeptide which retains at least
some of the immunogenicity or antigenicity of the source protein or
proteins. Fragments of multivalent oligopeptides, DTs, and/or PSMs
as described herein include proteolytic fragments, deletion
fragments or fragments that exhibit increased solubility during
expression, purification, and/or administration to an animal.
Fragments of multivalent oligopeptides, DTs, and/or PSMs as
described herein further include proteolytic fragments or deletion
fragments which exhibit reduced pathogenicity or toxicity when
delivered to a subject. Polypeptide fragments further include any
portion of the polypeptide which comprises an antigenic or
immunogenic epitope of the source polypeptide, including linear as
well as three-dimensional epitopes.
[0057] An "epitopic fragment" of a polypeptide is a portion of the
polypeptide that contains an epitope. An "epitopic fragment" can,
but need not, contain amino acid sequence in addition to one or
more epitopes.
[0058] The term "variant," as used herein, refers to a polypeptide
that differs from the recited polypeptide due to amino acid
substitutions, deletions, insertions, and/or modifications.
Non-naturally occurring variants can be produced using art-known
mutagenesis techniques. In some embodiments, variant polypeptides
differ from an identified sequence by substitution, deletion or
addition of three amino acids or fewer. Such variants can generally
be identified by modifying a polypeptide sequence, and evaluating
the antigenic or pathogenic properties of the modified polypeptide
using, for example, the representative procedures described herein.
In some embodiments, variants of a multivalent oligopeptide, DT,
and/or PSM form a protein complex which is less toxic than the
wild-type complex.
[0059] Polypeptide variants disclosed herein exhibit at least about
85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity
with identified polypeptide. Variant polypeptides can comprise
conservative or non-conservative amino acid substitutions,
deletions or insertions. Variants can comprise multivalent
oligopeptides, DTs, and/or PSMs identical to the various wild-type
staphylococcal proteins except for at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15, 20, or more amino acid substitutions, including specific
mutations described elsewhere herein, where the substitutions
render complex less toxic than a corresponding wild-type protein
complex. Derivatives of multivalent oligopeptides, DTs, and/or PSMs
as described herein are polypeptides which have been altered so as
to exhibit additional features not found on the native polypeptide.
Examples include fusion proteins. An analog is another form of a
multivalent oligopeptide, DT, and/or PSM described herein. An
example is a proprotein which can be activated by cleavage of the
proprotein to produce an active mature polypeptide.
[0060] Variants can also, or alternatively, contain other
modifications, whereby, for example, a polypeptide can be
conjugated or coupled, e.g., fused to a heterologous amino acid
sequence, e.g., a signal (or leader) sequence at the N-terminal end
of the protein which co-translationally or post-translationally
directs transfer of the protein. The polypeptide can also be
conjugated or produced coupled to a linker or other sequence for
ease of synthesis, purification or identification of the
polypeptide (e.g., 6-His), or to enhance binding of the polypeptide
to a solid support. For example, the polypeptide can be conjugated
or coupled to an immunoglobulin Fc region. The polypeptide can also
be conjugated or coupled to a sequence that imparts or modulates
the immune response to the polypeptide (e.g., a T-cell epitope,
B-cell epitope, cytokine, chemokine, etc.) and/or enhances uptake
and/or processing of the polypeptide by antigen presenting cells or
other immune system cells. The polypeptide can also be conjugated
or coupled to other polypeptides/epitopes from Staphylococcus sp.
and/or from other bacteria and/or other viruses to generate a
hybrid immunogenic protein that alone or in combination with
various adjuvants can elicit protective immunity to other
pathogenic organisms. The polypeptide can also be conjugated or
coupled to moieties which confer greater stability or improve half
life such as, but not limited to albumin, an immunoglobulin Fc
region, polyethylene glycol (PEG), and the like. The polypeptide
can also be conjugated or coupled to moieties (e.g., immunogenic
carbohydrates, e.g., a capsular polysaccharide or a surface
polysaccharide) from Staphylococcus sp. and/or from other bacteria
and/or other viruses to generate a modified immunogenic protein
that alone or in combination with one or more adjuvants can enhance
and/or synergize protective immunity. In certain embodiments, the
polypeptide described herein further comprises an immunogenic
carbohydrate. In one embodiment, the immunogenic carbohydrate is a
saccharide.
[0061] The term "saccharide" throughout this specification can
indicate polysaccharide or oligosaccharide and includes both.
Polysaccharides of the disclosure can be isolated from bacteria and
can be sized by known methods. For example, full length
polysaccharides can be "sized" (e.g., their size can be reduced by
various methods such as acid hydrolysis treatment, hydrogen
peroxide treatment, sizing by EMULSIFLEX.RTM. followed by a
hydrogen peroxide treatment to generate oligosaccharide fragments
or microfluidization). Polysaccharides can be sized in order to
reduce viscosity in polysaccharide samples and/or to improve
filterability for conjugated products. Oligosaccharides have a low
number of repeat units (e.g., 5-30 repeat units) and are typically
hydrolyzed polysaccharides. Polysaccharides of the disclosure can
be produced recombinantly.
[0062] S. aureus capsular antigens are surface associated, limited
in antigenic specificity, and highly conserved among clinical
isolates. In one embodiment, the immunogenic carbohydrate of the
disclosure is a capsular polysaccharide (CP) of S. aureus. In one
embodiment, a capsular saccharide can be a full length
polysaccharide, however in other embodiments it can be one
oligosaccharide unit, or a shorter than native length saccharide
chain of repeating oligosaccharide units. Serotyping studies of
staphylococcal isolates have revealed several putative capsular
serotypes, with types 5 and 8 (CP5 and CP8) being the most
prevalent among isolates from clinical infections, accounting for
about 25% and 50% of isolates recovered from humans, respectively
(O'Riordan and Lee, Clinical Microbiology Reviews, January 2004, p.
218-234, Vol. 17, No. 1; Poutrel and Sutra, J Clin Microbiol. 1993
February; 31(2):467-9). The same isolates were also recovered from
poultry, cows, horses and pigs (Tollersrud et al., J Clin
Microbiol. 2000 August; 38(8):2998-3003; Cunnion K M et al., Infect
Immun. 2001 November; 69(11):6796-803). Type 5 and 8 capsular
polysaccharides purified from the prototype strains Reynolds and
Becker, respectively, are structurally very similar to each other
and to the capsule made by strain T, described previously by Wu and
Park (Wu and Park. 1971. J. Bacteriol. 108:874-884). Type 5 has the
structure
(.fwdarw.4)-3-O-Ac-.beta.-D-ManNAcA-(1.fwdarw.4)-.omega.-L-FucNAc-(1.fwda-
rw.3)-.beta.-D-FucNAc-(1.fwdarw.).sub.n, (Fournier, J. M., et al.,
1987. Ann. Inst. Pasteur Microbiol. 138:561-567; Moreau. M., et
al., 1990. Carbohydr. Res. 201:285-297), and type 8 has the
structure
(.fwdarw.3)-4-O-Ac-.beta.-D-ManNAcA-(1.fwdarw.3)-.alpha.-L-FucNAc-(1.fwda-
rw.3)-.beta.-D-FucNAc-(1.fwdarw.).sub.n (Fournier, J. M., et al,
1984. Infect. Imnmun. 45:87-93). Type 5 and 8 polysaccharides
differ only in the linkages between the sugars and in the sites of
O-acetylation of the mannosaminuronic acid residues, yet they are
serologically distinct.
[0063] Type 5 and 8 CP conjugated to a detoxified recombinant
Pseudomonas aeruginosa exotoxin A carrier were shown to be highly
immunogenic and protective in a mouse model (A Fattom et al.,
Infect Immun. 1993 March; 61(3): 1023-1032; A Fattom et al., Infect
Immun. 1996 May; 64(5): 1659-1665) and passive transfer of the
CP5-specific antibodies from the immunized animals induced
protection against systemic infection in mice (Lee et al., Infect
Immun. 1997 October; 65(10): 4146-4151) and against endocarditis in
rats challenged with a serotype 5 S. aureus (Shinefield H et al., N
Engl J Med. 2002 Feb. 14; 346(7):491-6). A bivalent CP5 and CP8
conjugate vaccine (StaphVAX.RTM., Nabi Biopharmaceutical) was
developed that provided 75% protection in mice against S. aureus
challenge. The vaccine has been tested on humans (Fattom A I et
al., Vaccine. 2004 Feb. 17; 22(7):880-7; Maira-Litran T et al.,
Infect Immun. 2005 October; 73(10):6752-62). In certain
embodiments, the recombinant peptide or multivalent oligopeptide of
the disclosure is combined with or conjugated to an immunogenic
carbohydrate (e.g., CP5, CP8, a CP fragment or a combination
thereof).
[0064] Immunization with poly-N-acetylglucosamine (PNAG) (McKenney
D. et al., Science. 1999 May 28; 284(5419): 1523-7) or
poly-N-succinyl glucosamine (PNSG) (Tuchscherr L P. et al., Infect
Immun. 2008 December; 76(12):5738-44. Epub 2008 Sep. 22), both S.
aureus surface carbohydrates, has been shown to generate at least
partial protection against S. aureus challenge in experimental
animal models. PNSG was identified as the chemical form of the S.
epidermidis capsular polysaccharide/adhesin (PS/A) which mediates
adherence of coagulase-negative staphylococci (CONS) to
biomaterials, serves as the capsule for strains of CoNS that
express PS/A, and is a target for protective antibodies. PNSG is
also made by S. aureus, where it is an environmentally regulated,
in vivo-expressed surface polysaccharide and similarly serves as a
target for protective immunity (McKenney D. et al., J. Biotechnol.
2000 Sep. 29; 83(1-2): 37-44). In certain embodiments of the
disclosure, the immunogenic carbohydrate is a surface
polysaccharide, e.g., poly-N-acetylglucosamine (PNAG),
poly-N-succinyl glucosamine (PNSG), a surface polysaccharide
fragment or a combination thereof.
[0065] Wall Teichoic Acid (WTA) is a prominent polysaccharide
widely expressed on S. aureus strains (Neuhaus, F. C. and J.
Baddiley, Microbiol Mol Biol Rev, 2003. 67(4):686-723) and antisera
to WTA have been shown to induce opsonophagocytic killing alone and
in presence of complement ((Thakker, M., et al., Infect Immun,
1998. 66(11):5183-9), and Fattom et al, U.S. Pat. No. 7,754,225).
WTA is linked to peptidoglycans and protrudes through the cell wall
becoming prominently exposed on non-encapsulated strains such as
USA300 responsible for most cases of community acquired MRSA (CA
MRSA) in the US (Hidron, A. I., et al., Lancet Infect Dis, 2009.
9(6):384-92).
[0066] Lipoteichoic acid (LTA) is a constituent of the cell wall of
Gram-positive bacteria, e.g., Staphylococcus aureus. LTA can bind
to target cells non-specifically through membrane phospholipids, or
specifically to CD14 and to Toll-like receptors. Target-bound LTA
can interact with circulating antibodies and activate the
complement cascade to induce a passive immune kill phenomenon. It
also triggers the release from neutrophils and macrophages of
reactive oxygen and nitrogen species, acid hydrolases, highly
cationic proteinases, bactericidal cationic peptides, growth
factors, and cytotoxic cytokines, which can act in synergy to
amplify cell damage.
[0067] In certain embodiments, a surface polysaccharide is combined
with or conjugated to a polypeptide of the disclosure. In certain
embodiments the surface polysaccharide is, e.g.,
poly-N-acetylglucosamine (PNAG), poly-N-succinyl glucosamine
(PNSG), Wall Teichoic Acid (WTA), Lipoteichoic acid (LPA), a
fragment of any of said surface polysaccharides, or a combination
of two or more of said surface polysaccharides.
[0068] The term "sequence identity" as used herein refers to a
relationship between two or more polynucleotide sequences or
between two or more polypeptide sequences. When a position in one
sequence is occupied by the same nucleic acid base or amino acid in
the corresponding position of the comparator sequence, the
sequences are said to be "identical" at that position. The
percentage "sequence identity" is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid occurs in both sequences to yield the number of
"identical" positions. The number of "identical" positions is then
divided by the total number of positions in the comparison window
and multiplied by 100 to yield the percentage of "sequence
identity." Percentage of "sequence identity" is determined by
comparing two optimally aligned sequences over a comparison window
and a homologous polypeptide from another isolate. In order to
optimally align sequences for comparison, the portion of a
polynucleotide or polypeptide sequence in the comparison window can
comprise additions or deletions termed gaps while the reference
sequence is kept constant. An optimal alignment is that alignment
which, even with gaps, produces the greatest possible number of
"identical" positions between the reference and comparator
sequences. Percentage "sequence identity" between two sequences can
be determined using the version of the program "BLAST 2 Sequences"
which is available from the National Center for Biotechnology
Information as of Sep. 1, 2004, which program incorporates the
programs BLASTN (for nucleotide sequence comparison) and BLASTP
(for polypeptide sequence comparison), which programs are based on
the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA
90(12):5873-5877, 1993). When utilizing "BLAST 2 Sequences,"
parameters that were default parameters as of Sep. 1, 2004, can be
used for word size (3), open gap penalty (11), extension gap
penalty (1), gap drop-off (50), expect value (10) and any other
required parameter including but not limited to matrix option.
[0069] The term "epitope," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
for example a mammal, for example, a human. An "immunogenic
epitope," as used herein, is defined as a portion of a protein that
elicits an immune response in an animal, as determined by any
method known in the art. The term "antigenic epitope," as used
herein, is defined as a portion of a protein to which an antibody
or T-cell receptor can immunospecifically bind its antigen as
determined by any method well known in the art. Immunospecific
binding excludes non-specific binding but does not necessarily
exclude cross-reactivity with other antigens. Whereas all
immunogenic epitopes are antigenic, antigenic epitopes need not be
immunogenic.
[0070] As used herein, a "coding region" is a portion of nucleic
acid which consists of codons translated into amino acids. Although
a "stop codon" (TAG, TGA, or TAA) is not translated into an amino
acid, it can be considered to be part of a coding region, but any
flanking sequences, for example promoters, ribosome binding sites,
transcriptional terminators, and the like, are outside the coding
region.
[0071] The term "codon optimization" is defined herein as modifying
a nucleic acid sequence for enhanced expression in the cells of the
host of interest by replacing at least one, more than one, or a
significant number, of codons of the native sequence with codons
that are more frequently or most frequently used in the genes of
that host. Various species exhibit particular bias for certain
codons of a particular amino acid.
[0072] The terms "composition" or "pharmaceutical composition" can
include compositions containing immunogenic polypeptides of the
disclosure along with e.g., adjuvants or pharmaceutically
acceptable carriers, excipients, or diluents, which are
administered to an individual already suffering from S. aureus
infection or an individual in need of immunization against S.
aureus infection.
[0073] The term "pharmaceutically acceptable" refers to
compositions that are, within the scope of sound medical judgment,
suitable for contact with the tissues of human beings and animals
without excessive toxicity or other complications commensurate with
a reasonable benefit/risk ratio. In some embodiments, the
polypeptides, polynucleotides, compositions, and vaccines described
herein are pharmaceutically acceptable.
[0074] An "effective amount" is that amount the administration of
which to an individual, either in a single dose or as part of a
series, is effective for treatment or prevention. An amount is
effective, for example, when its administration results in a
reduced incidence of S. aureus infection relative to an untreated
individual, as determined, e.g., after infection or challenge with
infectious S. aureus, including, but is not limited to reduced
bacteremia, reduced toxemia, reduced sepsis, reduced symptoms,
increased immune response, modulated immune response, or reduced
time required for recovery. This amount varies depending upon the
health and physical condition of the individual to be treated, the
taxonomic group of individual to be treated (e.g., human, nonhuman
primate, primate, etc.), the responsive capacity of the
individual's immune system, the extent of treatment or protection
desired, the formulation of the vaccine, a professional assessment
of the medical situation, and other relevant factors. It is
expected that the effective amount will fall in a relatively broad
range that can be determined through routine trials. Typically a
single dose is from about 10 .mu.g to 10 mg/kg body weight of
purified polypeptide or an amount of a modified carrier organism or
virus, or a fragment or remnant thereof, sufficient to provide a
comparable quantity of recombinantly expressed multivalent
oligopeptide, DT, and/or PSM as described herein. The term "peptide
vaccine" or "subunit vaccine" refers to a composition comprising
one or more polypeptides described herein, which when administered
to an animal are useful in stimulating an immune response against
staphylococcal (e.g., S. aureus) infection.
[0075] The term "subject" is meant any subject, particularly a
mammalian subject, for whom diagnosis, prognosis, immunization, or
therapy is desired. Mammalian subjects include, but are not limited
to, humans, domestic animals, farm animals, zoo animals such as
bears, sport animals, pet animals such as dogs, cats, guinea pigs,
rabbits, rats, mice, horses, cattle, bears, cows; primates such as
apes, monkeys, orangutans, and chimpanzees; canids such as dogs and
wolves; felids such as cats, lions, and tigers; equids such as
horses, donkeys, and zebras; food animals such as cows, pigs, and
sheep; ungulates such as deer and giraffes; rodents such as mice,
rats, hamsters and guinea pigs; and so on. In one embodiment, the
subject is a human subject.
[0076] As used herein, a "subject in need thereof" refers to an
individual for whom it is desirable to treat, i.e., to prevent,
cure, retard, or reduce the severity of staphylococcal (e.g., S.
aureus) disease symptoms, or result in no worsening of disease
cause by S. aureus over a specified period of time, or both.
[0077] The terms "priming" or "primary" and "boost" or "boosting"
as used herein to refer to the initial and subsequent
immunizations, respectively, i.e., in accordance with the
definitions these terms normally have in immunology. However, in
certain embodiments, e.g., where the priming component and boosting
component are in a single formulation, initial and subsequent
immunizations are not be necessary as both the "prime" and the
"boost" compositions are administered simultaneously.
II. Delta Toxin and Phenol-Soluble Modulin Peptides and Multivalent
Oligopeptides
[0078] This disclosure provides recombinant oligopeptide fusion
proteins comprised of peptide subunits derived from staphylococcal
toxins and superantigens. In certain embodiments an oligopeptide as
provided herein comprises a mutant or wild-type delta toxin peptide
(DT) or a mutant or wild-type phenol-soluble modulin peptide (PSM).
Wild-type DT and the six forms of PSM are presented in Table 1.
TABLE-US-00001 TABLE 1 WILD-TYPE DT AND PSMS SEQ SEQUENCE ID NO
delta toxin WT MAQDIISTIGDLVKWIIDTVNKFTKK 1 PSM.alpha.1 WT
MGIIAGIIKVIKSLIEQFTGK 38 PSM.alpha.2 WT MGIIAGIIKFIKGLIEKFTGK 12
PSM.alpha.3 WT MEFVAKLFKFFKDLLGKFLGNN 13 PSM.alpha.4 WT
MAIVGTIIKIIKAIIDIFAK 14 PSM.beta.1 WT MEGLFNAIKDTVTAAINNDGAKLGTSIV
15 NIVENGVGLLSKLFGF PSM.beta.2 WT MTGLAEAIANTVQAAQQHDSVKLGTSIV 16
DIVANGVGLLGKLFGF PSM-mec MDFTGVITSI IDLIKTCIQA FG 52
[0079] In one aspect, the disclosure provides a recombinant peptide
comprising a Staphylococcus delta toxin peptide or a mutant,
fragment, variant or derivative thereof (DT); a Staphylococcus
phenol soluble modulin peptide or a mutant, fragment, variant or
derivative thereof (PSM); or a fusion of a DT and a PSM. A DT or
PSM as provided herein can be mutated to reduce toxicity, e.g.,
surfactant properties, while retaining antigenicity. Accordingly, a
recombinant DT, PSM, or both as provided by this disclosure can be
attenuated relative to wild-type DT, PSM, or both, and yet the
peptide can elicit an anti-Staphylococcus aureus immune response
when administered to a subject. The antigenicity of DT and PSM can
rely on maintenance of the peptide's alpha-helical structure, where
the surfactant properties can rely on the toxin having a
hydrophobic face. Thus in certain aspects, the disclosure provides
a DT, a PSM or both, where hydrophobicity of the peptide is reduced
relative to the wild-type peptide. For example, hydrophobicity can
be reduced by replacing at least one, e.g., one, two, three, four,
five or more hydrophobic amino acids which make up the hydrophobic
face of the toxin with less hydrophobic amino acids. Hydrophobic
amino acids include, but are not limited to valine (V), leucine
(L), isoleucine (I), phenylalanine (F), and methionine (M). In
certain aspects, a hydrophobic amino acid is replaced with a small
amino acid that would not be expected to alter the alpha helical
structure of the peptide. For example, the hydrophobic amino acid
can be replaced with alanine (A) or glycine (G).
[0080] In one aspect, the disclosure provides a recombinant peptide
comprising mutated DT, where the DT comprises the amino acid
sequence:
TABLE-US-00002 (SEQ ID NO: 39)
MAQDX.sub.5X.sub.6STX.sub.9GDX.sub.12X.sub.13KWX.sub.16X.sub.17DTX.sub.20-
NKFTKK
According to this aspect, at least one of X.sub.5, X.sub.6,
X.sub.9, X.sub.12, X.sub.13, X.sub.16, X.sub.17, or X.sub.20
comprises an amino acid substitution relative to SEQ ID NO: 1.
Thus, according to this aspect the DT is not wild-type DT.
According to this aspect, X.sub.5 can be isoleucine (I), glycine
(G) or alanine (A), X.sub.6 can be I, G, or A, X.sub.9 can be I, G,
or A, X.sub.12 can be leucine (L), G, or V, X.sub.13 can be valine
(V), G, or A, X.sub.16 can be I, G, or A, X.sub.17 can be I, G, or
A, and X.sub.20 can be V, G, or A. In certain aspects, only a
single amino acid from among X.sub.5, X.sub.6, X.sub.9, X.sub.12,
X.sub.13, X.sub.16, X.sub.17, or X.sub.20 is substituted relative
to SEQ ID NO: 1. Thus only one of X.sub.5, X.sub.6, X.sub.9,
X.sub.12, X.sub.13, X.sub.16, X.sub.17, or X.sub.20 is G or A. In
certain aspects X.sub.12 is the single substituted amino acid.
According to this aspect, X.sub.12 can be G, and the peptide
comprises the amino acid sequence SEQ ID NO: 2, or X.sub.12 can be
A and the peptide comprises the amino acid sequence SEQ ID NO: 4.
In another aspect, two amino acids from among X.sub.5, X.sub.6,
X.sub.9, X.sub.12, X.sub.13, X.sub.16, X.sub.17, or X.sub.20 are
substituted relative to SEQ ID NO: 2. For example, X.sub.12 and
X.sub.20 can be substituted, and can be G or A. Thus in certain
aspects X.sub.12 can independently be G or A, and X.sub.20 can
independently be G or A. For example, the DT can comprise the amino
acid sequence SEQ ID NO: 3, or the amino acid sequence SEQ ID NO:
5. Other amino acid substitutions, either single, or multiple
substitutions, can easily be visualized and constructed by a person
of ordinary skill in the art according to this disclosure.
[0081] In another aspect, the disclosure provides a recombinant
peptide comprising a mutated PSM, e.g., a mutant PSM.alpha.1,
PSM.alpha.2, PSM.alpha.3, PSM.alpha.4, PSM.beta.1, PSM.beta.2,
PSM-mec, or any combination of two or more PSMs.
[0082] In one aspect, the peptide comprises a mutant PSM.alpha.1
comprising the amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 40)
X.sub.1GX.sub.3X.sub.4AGX.sub.7X.sub.8KX.sub.10X.sub.11KSX.sub.14X.sub.15-
EQX.sub.18TGK
According to this aspect, at least one of X.sub.1, X.sub.3,
X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14, X.sub.15,
or X.sub.18 comprises an amino acid substitution relative to SEQ ID
NO: 38. Thus, according to this aspect the PSM.alpha.1 is not
wild-type PSM.alpha.1 According to this aspect, X.sub.1 can be
methionine (M), G, or A, X.sub.3 can be I, G, or A, X.sub.4 can be
I, G, or A, X.sub.7 can be I, G, or A, X.sub.8 can be I, G, or A,
X.sub.10 can be V, G, or A, X.sub.11 can be I, G, or A, X.sub.14
can be L, G, or A, X.sub.15 can be I, G, or A, and X.sub.18 can be
F, G, or A. In certain aspects, only a single amino acid from among
X.sub.1, X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11,
X.sub.14, X.sub.15, and X.sub.18 is substituted relative to SEQ ID
NO: 38. Thus only one of X.sub.1, X.sub.3, X.sub.4, X.sub.7,
X.sub.8, X.sub.10, X.sub.11, X.sub.14, X.sub.15, and X.sub.18 is G
or A. In another aspect, two amino acids from among X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, and X.sub.18 are substituted relative to SEQ ID NO: 38.
Various amino acid substitutions in PSM.alpha.1, either single, or
multiple substitutions, can easily be visualized and constructed by
a person of ordinary skill in the art according to this
disclosure.
[0083] In one aspect, the peptide comprises a mutant PSM.alpha.2
comprising the amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 41)
X.sub.1GX.sub.3X.sub.4AGX.sub.7X.sub.8KX.sub.10X.sub.11KGX.sub.14X.sub.15-
EKX.sub.18TGK
According to this aspect, at least one of X.sub.1, X.sub.3,
X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14, X.sub.15,
or X.sub.18 comprises an amino acid substitution relative to SEQ ID
NO: 12. Thus, according to this aspect the PSM.alpha.2 is not
wild-type PSM.alpha.2. According to this aspect, X.sub.1 can be M,
G, or A, X.sub.3 can be I, G, or A, X.sub.4 can be I, G, or A,
X.sub.7 can be I, G, or A, X.sub.8 can be I, G, or A, X.sub.10 can
be F, G, or A, X.sub.11 can be I, G, or A, X.sub.14 can be L, G, or
A, X.sub.15 can be I, G, or A, and X.sub.18 can be F, G, or A. In
certain aspects, only a single amino acid from among X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, and X.sub.18 is substituted relative to SEQ ID NO: 12.
Thus only one of X.sub.1, X.sub.3, X.sub.4, X.sub.7, X.sub.8,
X.sub.10, X.sub.11, X.sub.14, X.sub.15, and X.sub.18 is G or A. In
another aspect, two amino acids from among X.sub.1, X.sub.3,
X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14, X.sub.15,
and X.sub.18 are substituted relative to SEQ ID NO: 12. Various
amino acid substitutions, either single, or multiple substitutions,
can easily be visualized and constructed by a person of ordinary
skill in the art according to this disclosure.
[0084] In one aspect, the peptide comprises a mutant PSM.alpha.3
comprising the amino acid sequence:
TABLE-US-00005 (SEQ ID NO: 42)
X.sub.1EX.sub.3X.sub.4AKX.sub.7X.sub.8KX.sub.10X.sub.11KDX.sub.14X.sub.15-
GKX.sub.18X.sub.19GNN
According to this aspect, at least one of X.sub.1, X.sub.3,
X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14, X.sub.15,
X.sub.18, or X.sub.19 comprises an amino acid substitution relative
to SEQ ID NO: 6. Thus, according to this aspect the PSM.alpha.3 is
not wild-type PSM.alpha.3. According to this aspect, X.sub.1 can be
M, G, or A, X.sub.3 can be F, G, or A, X.sub.4 can be V, G, or A,
X.sub.7 can be L, G, or A, X.sub.8 can be F, G, or A, X.sub.10 can
be F, G, or A, X.sub.1 can be F, G, or A, X.sub.14 can be L, G, or
A, X.sub.15 can be L, G, or A, X.sub.18 can be F, G, or A, and
X.sub.19 can be L, G, or A. In certain aspects, only a single amino
acid from among X.sub.1, X.sub.3, X.sub.4, X.sub.7, X.sub.8,
X.sub.10, X.sub.11, X.sub.14, X.sub.15, X.sub.18, and X.sub.19 is
substituted relative to SEQ ID NO: 6. Thus only one of X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, X.sub.18, and X.sub.19 is G or A. In certain aspects
X.sub.14 is the single substituted amino acid. According to this
aspect, X.sub.14 can be G, and the peptide comprises the amino acid
sequence SEQ ID NO: 9, or X.sub.14 can be A and the peptide
comprises the amino acid sequence SEQ ID NO: 7. In another aspect,
two amino acids from among X.sub.1, X.sub.3, X.sub.4, X.sub.7,
X.sub.8, X.sub.10, X.sub.11, X.sub.14, X.sub.15, X.sub.18, and
X.sub.19 are substituted relative to SEQ ID NO: 6. For example,
X.sub.4 and X.sub.14 can be substituted, and can be G or A. Thus in
certain aspects X.sub.4 can independently be G or A, and X.sub.14
can independently be G or A. For example, the PSM.alpha.3 can
comprise the amino acid sequence SEQ ID NO: 8, the amino acid
sequence SEQ ID NO: 10, or the amino acid sequence SEQ ID NO: 11.
Other amino acid substitutions, either single, or multiple
substitutions, can easily be visualized and constructed by a person
of ordinary skill in the art according to this disclosure.
[0085] In one aspect, the peptide comprises a mutant PSM.alpha.4
comprising the amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 43)
X.sub.1AX.sub.3X.sub.4GTX.sub.7X.sub.8KX.sub.10X.sub.11KAX.sub.14X.sub.15-
DX.sub.17X.sub.18AK
According to this aspect, at least one of X.sub.1, X.sub.3,
X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14, X.sub.15,
X.sub.17, or X.sub.18 comprises an amino acid substitution relative
to SEQ ID NO: 14. Thus, according to this aspect the PSM.alpha.4 is
not wild-type PSM.alpha.4. According to this aspect, X.sub.1 can be
M, G, or A, X.sub.3 can be I, G, or A, X.sub.4 can be V, G, or A,
X.sub.7 can be I, G, or A, X.sub.8 can be I, G, or A, X.sub.10 can
be I, G, or A, X.sub.11 can be I, G, or A, X.sub.14 can be I, G, or
A, X.sub.15 can be I, G, or A, X.sub.17 can be I, G, or A, and
X.sub.18 can be F, G, or A. In certain aspects, only a single amino
acid from among X.sub.1, X.sub.3, X.sub.4, X.sub.7, X.sub.8,
X.sub.10, X.sub.11, X.sub.14, X.sub.15, X.sub.17, and X.sub.18 is
substituted relative to SEQ ID NO: 14. Thus only one of X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, X.sub.17, and X.sub.18 is G or A. In another aspect, two
amino acids from among X.sub.1, X.sub.3, X.sub.4, X.sub.7, X.sub.8,
X.sub.10, X.sub.11, X.sub.14, X.sub.15, X.sub.17, and X.sub.18 are
substituted relative to SEQ ID NO: 14. Other amino acid
substitutions, either single, or multiple substitutions, can easily
be visualized and constructed by a person of ordinary skill in the
art according to this disclosure.
[0086] In one aspect, the peptide comprises a mutant PSM.beta.1
comprising the amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 44)
MEGX.sub.4X.sub.5NAX.sub.8KDTX.sub.12TAAX.sub.16NNDGAKLGTSIVNIVENGVGLLSKLF-
GF
According to this aspect, at least one of X.sub.4, X.sub.5,
X.sub.8, X.sub.12, or X.sub.16 comprises an amino acid substitution
relative to SEQ ID NO: 15. Thus, according to this aspect the
PSM.beta.1 is not wild-type PSM.beta.1. According to this aspect,
X.sub.4 can be L, G, or A, X.sub.5 can be F, G, or A, X.sub.8 can
be I, G, or A, X.sub.12 can be V, G, or A, and X.sub.16 can be I,
G, or A. In certain aspects, only a single amino acid from among
X.sub.4, X.sub.5, X.sub.8, X.sub.12, and X.sub.16 is substituted
relative to SEQ ID NO: 15. Thus only one of X.sub.4, X.sub.5,
X.sub.8, X.sub.12, and X.sub.16 is G or A. In another aspect, two
amino acids from among X.sub.4, X.sub.5, X.sub.8, X.sub.12, and
X.sub.16 are substituted relative to SEQ ID NO: 15. Other amino
acid substitutions, either single, or multiple substitutions, can
easily be visualized and constructed by a person of ordinary skill
in the art according to this disclosure.
[0087] In one aspect, the peptide comprises a mutant PSM.beta.2
comprising the amino acid sequence:
TABLE-US-00008 (SEQ ID NO: 45)
MTGX.sub.4AEAX.sub.8ANTX.sub.12QAAQQHDSVKX.sub.23GTSIVDIVANGVGLLGKLFGF
According to this aspect, at least one of X.sub.4, X.sub.8,
X.sub.12, or X.sub.23 comprises an amino acid substitution relative
to SEQ ID NO: 16. Thus, according to this aspect the PSM.beta.2 is
not wild-type PSM.beta.2. According to this aspect, X.sub.4 can be
L, G, or A, X.sub.8 can be I, G, or A, X.sub.12 can be V, G, or A,
and X.sub.23 can be L, G, or A. In certain aspects, only a single
amino acid from among X.sub.4, X.sub.8, X.sub.12, and X.sub.23 is
substituted relative to SEQ ID NO: 16. Thus only one of X.sub.4,
X.sub.8, X.sub.12, and X.sub.23 is G or A. In another aspect, two
amino acids from among X.sub.4, X.sub.8, X.sub.12, and X.sub.23 are
substituted relative to SEQ ID NO: 16. Other amino acid
substitutions, either single, or multiple substitutions, can easily
be visualized and constructed by a person of ordinary skill in the
art according to this disclosure.
[0088] In one aspect, the peptide comprises a mutant PSM-mec
comprising the amino acid sequence:
TABLE-US-00009 (SEQ ID NO: 53)
MDX.sub.3TGX.sub.6X.sub.7TSX.sub.10X.sub.11DX.sub.13X.sub.14KTX.sub.17X.s-
ub.18QAFG
According to this aspect, at least one of X.sub.3, X.sub.6,
X.sub.7, X.sub.10, X.sub.11, X.sub.13, X.sub.14, X.sub.17, or
X.sub.18 comprises an amino acid substitution relative to SEQ ID
NO: 52. Thus, according to this aspect the PSM-mec is not wild-type
PSM-mec. According to this aspect, X.sub.3 can be F, G, or A,
X.sub.6 can be V, G, or A, X.sub.7 can be I, G, or A, X.sub.10 can
be I, G, or A, X.sub.11 can be I, G, or A, X.sub.13 can be L, G, or
A, X.sub.14 can be I, G, or A, X.sub.17 can be C, G, or A, and
X.sub.18 can be I, G, or A. In certain aspects, only a single amino
acid from among X.sub.3, X.sub.6, X.sub.7, X.sub.10, X.sub.11,
X.sub.13, X.sub.14, X.sub.17, or X.sub.18 is substituted relative
to SEQ ID NO: 52. Thus only one of X.sub.3, X.sub.6, X.sub.7,
X.sub.10, X.sub.11, X.sub.13, X.sub.14, X.sub.17, or X.sub.18 is G
or A. In another aspect, two amino acids from among X.sub.1,
X.sub.3, X.sub.4, X.sub.7, X.sub.8, X.sub.10, X.sub.11, X.sub.14,
X.sub.15, and X.sub.18 are substituted relative to SEQ ID NO: 52.
Various amino acid substitutions in PSM-mec, either single, or
multiple substitutions, can easily be visualized and constructed by
a person of ordinary skill in the art according to this
disclosure.
[0089] The disclosure further provides a multivalent oligopeptide
comprising a fusion of two or more, e.g., two, three, four, five,
six, seven, eight, nine, ten or more Staphylococcus aureus-derived
peptides, or mutants, fragments, variants, or derivatives thereof
arranged in any order. The two or more Staphylococcus
aureus-derived peptides, or mutants, fragments, variants, or
derivatives thereof can be the same or different. A multivalent
oligopeptide as provided herein can include, without limitation,
two or more of:
[0090] a. a wild-type DT, a mutant DT as described above, or any
fragment, variant, or derivative thereof;
[0091] b. a wild-type PSM, a mutant PSM as described above, or any
fragment, variant, or derivative thereof;
[0092] c. an alpha hemolysin polypeptide or mutant, fragment,
variant, or derivative thereof, including without limitation AT-62
(SEQ ID NO: 46), or other alpha hemolysin peptides as described
elsewhere herein and in PCT Publication No. WO 2012/109167A1;
[0093] d. a leukocidin polypeptide or mutant, fragment, variant, or
derivative thereof, including, without limitation, a
Panton-Valentine leukocidin (PVL) LukS-PV subunit comprising an
amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NO: 47, a Panton-Valentine
leukocidin (PVL) LukF-PV subunit comprising an amino acid sequence
at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO: 48, or a combination thereof, or other leukocidin
peptides as described elsewhere herein and in PCT Publication No.
WO 2013/082558, including, without limitation, LukS-Mut9 (SEQ ID
NO: 54) and/or LukF-Mut-1 (SEQ ID NO: 55); or
[0094] e. a superantigen (SAg) polypeptide, or mutant, fragment,
variant, or derivative thereof.
[0095] In some embodiments, the peptides comprising the multivalent
oligopeptide can be directly fused to each other. In other
embodiments, the peptides comprising the multivalent oligopeptide
can be associated via a peptide linker. Suitable peptide linkers
can be chosen based on their ability to adopt a flexible, extended
conformation, or a secondary structure that can interact with
joined epitopes, or based on their ability to increase overall
solubility of the fusion polypeptide, or based on their lack of
electrostatic or water-interaction effects that influence joined
peptide regions. In certain aspects, a linker for use in a
multivalent oligopeptide as provided herein can include at least
one, but no more than 50 amino acids, e.g., small amino acids that
provide a flexible chain, e.g., glycine, serine, alanine, or a
combination thereof. In certain aspects, a linker for use in a
multivalent oligopeptide as provided herein can include
(GGGS).sub.n (SEQ ID NO: 56) or (GGGGS).sub.n (SEQ ID NO: 57),
wherein n is a integer from 1 to 10.
[0096] In certain aspects, the multivalent oligopeptide includes
AT-62 and DT, in any order, where AT-62 and DT can be fused
together via a linker sequence. In certain aspects, the multivalent
oligopeptide comprises, consists of, or consists essentially of
AT62_DT (SEQ ID NO: 18), or an oligopeptide comprising, consisting,
or consisting essentially of an amino acid sequence at least 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
18.
[0097] In certain aspects, the multivalent oligopeptide includes
AT-62 and a PSM, in any order, where AT-62 and PSM can be fused
together via a linker sequence. In certain aspects, the multivalent
oligopeptide comprises, consists of, or consists essentially of
AT62_PSM (SEQ ID NO: 20), or an oligopeptide comprising,
consisting, or consisting essentially of an amino acid sequence at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to
SEQ ID NO: 20.
[0098] In certain aspects, the multivalent oligopeptide includes
AT-62, DT, and a PSM, in any order, where AT-62, DT, and PSM can be
fused together via linker sequences. In certain aspects, the
multivalent oligopeptide comprises, consists of, or consists
essentially of AT62_DT_PSM (SEQ ID NO: 22), or an oligopeptide
comprising, consisting, or consisting essentially of an amino acid
sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to SEQ ID NO: 22.
[0099] In certain aspects, the multivalent oligopeptide includes
AT-62 and a recombinant SEB or mutant, fragment, variant, or
derivative thereof, in any order, where AT-62 and SEB can be fused
together via a linker sequence. In certain aspects, the multivalent
oligopeptide comprises, consists of, or consists essentially of
AT62_rSEB (SEQ ID NO: 23), or an oligopeptide comprising,
consisting, or consisting essentially of an amino acid sequence at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to
SEQ ID NO: 23. In certain aspects, the multivalent oligopeptide
comprises, consists of, or consists essentially of rSEB_AT62 (SEQ
ID NO: 26), or an oligopeptide comprising, consisting, or
consisting essentially of an amino acid sequence at least 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
26.
[0100] In certain aspects, the multivalent oligopeptide includes
AT-62, a recombinant SEB or mutant, fragment, variant, or
derivative thereof, and DT, in any order, where AT-62, SEB, and DT
can be fused together via a linker sequence. In certain aspects,
the multivalent oligopeptide comprises, consists of, or consists
essentially of AT62_rSEB_DT (SEQ ID NO: 29), or an oligopeptide
comprising, consisting, or consisting essentially of an amino acid
sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to SEQ ID NO: 29.
[0101] Where the provided oligopeptide includes a staphylococcal
SAg or mutant, fragment, variant, or derivative thereof, the SAg,
can include, without limitation, SEB, SEC1-3, SEE, SEH, SEI, SEK,
TSST-1, SpeC, SED, SpeA, or any mutant, fragment, variant, or
derivative thereof, or any combination thereof, in any order. In
certain aspects, the oligopeptide includes a staphylococcal
enterotoxin B (SEB) or mutant, fragment, variant, or derivative
thereof. In certain aspects, the SEB mutant is the attenuated
toxoid SEB.sub.L45R/Y89A/Y94A (SEQ ID NO: 49), or a polypeptide
comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 49. In certain
aspects, the oligopeptide includes a staphylococcal enterotoxin A
(SEA) or mutant, fragment, variant, or derivative thereof. In
certain aspects, the SEA mutant is the attenuated toxoid
SEA.sub.L48R/D70R/Y92A (SEQ ID NO: 50), or a polypeptide comprising
an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NO: 50. In certain aspects, the
oligopeptide includes a staphylococcal toxic shock syndrome toxin-1
(TSST-1) or mutant, fragment, variant, or derivative thereof. In
certain aspects, the TSST-1 mutant is the attenuated toxoid
TSST-1.sub.L30R/D27A/I46A (SEQ ID NO: 51), or a polypeptide
comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 51.
[0102] The SAg toxoids can be linked together in any order, either
with our without linkers. In certain aspects, the multivalent
oligopeptide includes SEB.sub.L45R/Y89A/Y94A ("B"),
SEA.sub.L48R/D70R/Y92A ("A"), and TSST-1.sub.L30R/D27A/I46A ("T"),
in any order, where the toxoids can be fused together via linker
sequences. In certain aspects, the multivalent oligopeptide
comprises, consists of, or consists essentially of a "BAT" fusion
(SEQ ID NO: 32), or an oligopeptide comprising, consisting, or
consisting essentially of an amino acid sequence at least 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In
certain aspects, the multivalent oligopeptide comprises, consists
of, or consists essentially of a "BTA" fusion (SEQ ID NO: 33), or
an oligopeptide comprising, consisting, or consisting essentially
of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% identical to SEQ ID NO: 33. In certain aspects,
the multivalent oligopeptide comprises, consists of, or consists
essentially of a "ABT" fusion (SEQ ID NO: 34), or an oligopeptide
comprising, consisting, or consisting essentially of an amino acid
sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to SEQ ID NO: 34. In certain aspects, the multivalent
oligopeptide comprises, consists of, or consists essentially of a
"ATB" fusion (SEQ ID NO: 35), or an oligopeptide comprising,
consisting, or consisting essentially of an amino acid sequence at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to
SEQ ID NO: 35. In certain aspects, the multivalent oligopeptide
comprises, consists of, or consists essentially of a "TAB" fusion
(SEQ ID NO: 36), or an oligopeptide comprising, consisting, or
consisting essentially of an amino acid sequence at least 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36. In
certain aspects, the multivalent oligopeptide comprises, consists
of, or consists essentially of a "TBA" fusion (SEQ ID NO: 37), or
an oligopeptide comprising, consisting, or consisting essentially
of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% identical to SEQ ID NO: 37.
[0103] In certain aspects the multivalent oligopeptide comprises,
consists of, or consists essentially of the amino acid sequence SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO. 34, SEQ
ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or any combination
thereof.
[0104] In another embodiment, the multivalent oligopeptide, DT,
and/or PSM as described herein, can be attached to a heterologous
polypeptide. Various heterologous polypeptides can be used,
including, but not limited to an N- or C-terminal peptide imparting
stabilization, secretion, or simplified purification, such as a
hexa-Histidine-tag, a ubiquitin tag, a NusA tag, a chitin binding
domain, ompT, ompA, pelB, DsbA, DsbC, c-myc, KSI, polyaspartic
acid, (Ala-Trp-Trp-Pro)n, polyphenyalanine, polycysteine,
polyarginine, a B-tag, a HSB-tag, green fluorescent protein (GFP),
influenza virus hemagglutinin (HAI), a calmodulin binding protein
(CBP), a galactose-binding protein, a maltose binding protein
(MBP), a cellulose binding domains (CBD's), dihydrofolate reductase
(DHFR), glutathione-S-transferase (GST), streptococcal protein G,
staphylococcal protein A, T7gene10, an
avidin/streptavidin/Strep-tag complex, trpE, chloramphenicol
acetyltransferase, lacZ (.beta.-Galactosidase), His-patch
thioredoxin, thioredoxin, a FLAG.TM. peptide (Sigma-Aldrich), an
S-tag, or a T7-tag. See, e.g., Stevens, R. C., Structure,
8:R177-R185 (2000). Heterologous polypeptides can also include any
pre- and/or pro-sequences that facilitate the transport,
translocations, processing and/or purification of a multivalent
oligopeptide, DT, and/or PSM as described herein from a host cell
or any useful immunogenic sequence, including but not limited to
sequences that encode a T-cell epitope of a microbial pathogen, or
other immunogenic proteins and/or epitopes.
[0105] In some embodiments, the multivalent oligopeptide, DT,
and/or PSM attached to a heterologous polypeptide, as described
herein, can include a peptide linker sequence joining sequences
that comprise two or more peptide regions. Suitable peptide linker
sequences can be chosen based on their ability to adopt a flexible,
extended conformation, or a secondary structure that could interact
with joined epitopes, or based on their ability to increase overall
solubility of the fusion polypeptide, or based on their lack of
electrostatic or water-interaction effects that influence joined
peptide regions.
[0106] In some embodiments, the multivalent oligopeptide, DT,
and/or PSM as described herein, is isolated. An "isolated"
polypeptide is one that has been removed from its natural milieu.
The term "isolated" does not connote any particular level of
purification. Recombinantly produced multivalent oligopeptides,
DTs, and/or PSMs as described herein, expressed in non-native host
cells is considered isolated for purposes of the disclosure, as is
the polypeptide which have been separated, fractionated, or
partially or substantially purified by any suitable technique,
including by filtration, chromatography, centrifugation, and the
like.
[0107] Production of multivalent oligopeptides, DTs, and/or PSMs as
described herein, can be achieved by culturing a host cell
comprising a polynucleotide which operably encodes the polypeptide
of the disclosure, and recovering the polypeptide. Determining
conditions for culturing such a host cell and expressing the
polynucleotide are generally specific to the host cell and the
expression system and are within the knowledge of one of skill in
the art. Likewise, appropriate methods for recovering the
polypeptide of the disclosure are known to those in the art, and
include, but are not limited to, chromatography, filtration,
precipitation, or centrifugation.
III. Polynucleotides
[0108] Also disclosed is an isolated polynucleotide comprising a
nucleic acid encoding a multivalent oligopeptide, DT, and/or PSM as
described elsewhere herein.
[0109] In certain embodiments, the isolated polynucleotide as
described herein further comprises non-coding regions such as
promoters, operators, or transcription terminators as described
elsewhere herein. In some embodiments, the disclosure is directed
to the polynucleotide as described herein, and further comprising a
heterologous nucleic acid. The heterologous nucleic acid can, in
some embodiments, encode a heterologous polypeptide fused to the
polypeptide as described herein. For example, the isolated
polynucleotide as described herein can comprise additional coding
regions encoding, e.g., a heterologous polypeptide fused to the
polypeptide as described herein, or coding regions encoding
heterologous polypeptides separate from the polypeptide as
described herein such as, but not limited to, selectable markers,
additional immunogens, immune enhancers, and the like.
[0110] Also provided are expression constructs, vectors, and/or
host cells comprising the polynucleotides described herein. An
example of an isolated polynucleotide is a recombinant
polynucleotide contained in a vector. Further examples of an
isolated polynucleotide include recombinant polynucleotides
maintained in heterologous host cells or purified (partially or
substantially) polynucleotides in solution. In certain embodiments
of the disclosure a polynucleotide is "recombinant." Isolated
polynucleotides or nucleic acids according to the disclosure
further include such molecules produced synthetically. The relative
degree of purity of a polynucleotide or polypeptide described
herein is easily determined by well-known methods.
[0111] Also included within the scope of the disclosure are
genetically engineered polynucleotides encoding the multivalent
oligopeptides, DTs, and/or PSMs as described herein. Modifications
of nucleic acids encoding the multivalent oligopeptides, DTs,
and/or PSMs as described herein, can readily be accomplished by
those skilled in the art, for example, by oligonucleotide-directed
site-specific mutagenesis or de novo nucleic acid synthesis.
[0112] Some embodiments disclose an isolated polynucleotide
comprising a nucleic acid that encodes a multivalent oligopeptide,
DT, and/or PSM as described herein, where the coding region
encoding the polypeptide has been codon-optimized. As appreciated
by one of ordinary skill in the art, various nucleic acid coding
regions will encode the same polypeptide due to the redundancy of
the genetic code. Deviations in the nucleotide sequence that
comprise the codons encoding the amino acids of any polypeptide
chain allow for variations in the sequence of the coding region.
Since each codon consists of three nucleotides, and the nucleotides
comprising DNA are restricted to four specific bases, there are 64
possible combinations of nucleotides, 61 of which encode amino
acids (the remaining three codons encode signals ending
translation). The "genetic code" which shows which codons encode
which amino acids is reproduced herein as Table 2. As a result,
many amino acids are designated by more than one codon. For
example, the amino acids alanine and proline are coded for by four
triplets, serine and arginine by six, whereas tryptophan and
methionine are coded by just one triplet. This degeneracy allows
for DNA base composition to vary over a wide range without altering
the amino acid sequence of the polypeptides encoded by the DNA.
TABLE-US-00010 TABLE 2 The Standard Genetic Code T C A G T TTT Phe
(F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC
Tyr (Y) TGC TTA Leu (L) TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG
Ser (S) TAG Ter TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H)
CGT Arg (R) CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu
(L) CCA Pro (P) CAA Gln (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG
Gln (Q) CGG Arg (R) A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser
(S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I) ACA
Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACG Thr (T) AAG Lys (K)
AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC
Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A)
GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly
(G)
[0113] It is to be appreciated that any polynucleotide that encodes
a polypeptide in accordance with the disclosure falls within the
scope of this disclosure, regardless of the codons used.
[0114] Many organisms display a bias for use of particular codons
to code for insertion of a particular amino acid in a growing
polypeptide chain. Codon preference or codon bias, differences in
codon usage between organisms, is afforded by degeneracy of the
genetic code, and is well documented among many organisms.
[0115] Different factors have been proposed to contribute to codon
usage preference, including translational selection, GC
composition, strand-specific mutational bias, amino acid
conservation, protein hydropathy, transcriptional selection and
even RNA stability. One factor that determines codon usage is
mutational bias that shapes genome GC composition. This factor is
most significant in genomes with extreme base composition: species
with high GC content (e.g., gram positive bacteria). Mutational
bias is responsible not only for intergenetic difference in codon
usage but also for codon usage bias within the same genome
(Ermolaeva M, Curr. Issues Mol. Biol. 3(4):91-97, 2001).
[0116] Codon bias often correlates with the efficiency of
translation of messenger RNA (mRNA), which is in turn believed to
be dependent on, inter alia, the properties of the codons being
translated and the availability of particular transfer RNA (tRNA)
molecules. The predominance of selected tRNAs in a cell is
generally a reflection of the codons used most frequently in
peptide synthesis. Accordingly, genes can be tailored for optimal
gene expression in a given organism based on codon
optimization.
[0117] The present disclosure provides a polynucleotide comprising
a codon-optimized coding region which encodes a multivalent
oligopeptide, DT, and/or PSM as described herein. The codon usage
is adapted for optimized expression in a given prokaryotic or
eukaryotic host cell. In certain aspects the codon usage is adapted
for optimized expression in E. coli.
[0118] In certain aspects an isolated polynucleotide is provided
comprising the nucleic acid sequence SEQ ID NO: 17, encoding
AT62_DT and optimized for expression in E. coli. In certain aspects
an isolated polynucleotide is provided comprising the nucleic acid
sequence SEQ ID NO: 19, encoding AT62_PSM and optimized for
expression in E. coli. In certain aspects an isolated
polynucleotide is provided comprising the nucleic acid sequence SEQ
ID NO: 21, encoding AT62_DT_PSM and optimized for expression in E.
coli. In certain aspects an isolated polynucleotide is provided
comprising the nucleic acid sequence SEQ ID NO: 25, encoding
AT62_rSEB and optimized for expression in E. coli. In certain
aspects an isolated polynucleotide is provided comprising the
nucleic acid sequence SEQ ID NO: 28, encoding rSEB_AT62 and
optimized for expression in E. coli. In certain aspects an isolated
polynucleotide is provided comprising the nucleic acid sequence SEQ
ID NO: 31, encoding AT62_rSEB_DT and optimized for expression in E.
coli.
[0119] Codon-optimized polynucleotides are prepared by
incorporating codons preferred for use in the genes of a given
species into the DNA sequence. Also provided are polynucleotide
expression constructs, vectors, host cells comprising
polynucleotides comprising codon-optimized coding regions which
encode a multivalent oligopeptide, DT, and/or PSM as described
herein.
[0120] Given the large number of gene sequences available for a
wide variety of animal, plant and microbial species, it is possible
to calculate the relative frequencies of codon usage. Codon usage
tables are readily available, for example, at the "Codon Usage
Database" available on the world wide web at
kazusa.or.jp/codon/(visited Oct. 12, 2011), and these tables can be
adapted in a number of ways. (Nakamura, Y., et al., "Codon usage
tabulated from the international DNA sequence databases: status for
the year 2000" Nucl. Acids Res. 28:292, 2000).
[0121] By utilizing available tables, one of ordinary skill in the
art can apply the frequencies to any given polypeptide sequence,
and produce a nucleic acid fragment of a codon-optimized coding
region which encodes a desired polypeptide, but which uses codons
optimal for a given species. For example, in some embodiments of
the disclosure, the coding region is codon-optimized for expression
in E. coli.
[0122] A number of options are available for synthesizing codon
optimized coding regions designed by any of the methods described
above, using standard and routine molecular biological
manipulations well known to those of ordinary skill in the art. In
addition, gene synthesis is readily available commercially.
IV. Vectors and Expression Systems
[0123] Further disclosed is a vector comprising the polynucleotide
as described herein. The term "vector," as used herein, refers to
e.g., any of a number of nucleic acids into which a desired
sequence can be inserted, e.g., by restriction and ligation, for
transport between different genetic environments or for expression
in a host cell. Nucleic acid vectors can be DNA or RNA. Vectors
include, but are not limited to, plasmids, phage, phagemids,
bacterial genomes, and virus genomes. A cloning vector is one which
is able to replicate in a host cell, and which is further
characterized by one or more endonuclease restriction sites at
which the vector can be cut in a determinable fashion and into
which a desired DNA sequence can be ligated such that the new
recombinant vector retains its ability to replicate in the host
cell. In the case of plasmids, replication of the desired sequence
can occur many times as the plasmid increases in copy number within
the host bacterium or just a single time per host before the host
reproduces by mitosis. In the case of phage, replication can occur
actively during a lytic phase or passively during a lysogenic
phase. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced. Other vectors are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome.
[0124] Any of a wide variety of suitable cloning vectors are known
in the art and commercially available which can be used with
appropriate hosts. As used herein, the term "plasmid" refers to a
circular, double-stranded construct made up of genetic material
(i.e., nucleic acids), in which the genetic material is
extrachromosomal and in some instances, replicates autonomously. A
polynucleotide described herein can be in a circular or linearized
plasmid or in any other sort of vector. Procedures for inserting a
nucleotide sequence into a vector, e.g., an expression vector, and
transforming or transfecting into an appropriate host cell and
cultivating under conditions suitable for expression are generally
known in the art.
[0125] The disclosure further provides a vector comprising a
nucleic acid sequence encoding a multivalent oligopeptide, DT,
and/or PSM as described herein. In certain embodiments the vector
is an expression vector capable of expressing the multivalent
oligopeptide, DT, and/or PSM as described herein in a suitable host
cell. The term "expression vector" refers to a vector that is
capable of expressing the polypeptide described herein, i.e., the
vector sequence contains the regulatory sequences required for
transcription and translation of a polypeptide, including, but not
limited to promoters, operators, transcription termination sites,
ribosome binding sites, and the like. The term "expression" refers
to the biological production of a product encoded by a coding
sequence. In most cases a DNA sequence, including the coding
sequence, is transcribed to form a messenger-RNA (mRNA). The
messenger-RNA is then translated to form a polypeptide product
which has a relevant biological activity. Also, the process of
expression can involve further processing steps to the RNA product
of transcription, such as splicing to remove introns, and/or
post-translational processing of a polypeptide product.
[0126] Vector-host systems include, but are not limited to, systems
such as bacterial, mammalian, yeast, insect or plant cell systems,
either in vivo, e.g., in an animal or in vitro, e.g., in bacteria
or in cell cultures. The selection of an appropriate host is deemed
to be within the scope of those skilled in the art from the
teachings herein. In certain embodiments, the host cell is a
bacterium, e.g., E. coli.
[0127] Host cells are genetically engineered (infected, transduced,
transformed, or transfected) with vectors of the disclosure. Thus,
one aspect of the disclosure is directed to a host cell comprising
a vector which contains the polynucleotide as describe herein. The
engineered host cell can be cultured in conventional nutrient media
modified as appropriate for activating promoters, selecting
transformants or amplifying the polynucleotides. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to the ordinarily skilled artisan. The term
"transfect," as used herein, refers to any procedure whereby
eukaryotic cells are induced to accept and incorporate into their
genome isolated DNA, including but not limited to DNA in the form
of a plasmid. The term "transform," as used herein, refers to any
procedure whereby bacterial cells are induced to accept and
incorporate into their genome isolated DNA, including but not
limited to DNA in the form of a plasmid.
[0128] Bacterial host-expression vector systems include, but are
not limited to, a prokaryote (e.g., E. coli), transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. In some
embodiments, the plasmids used with E. coli use the T7
promoter-driven system regulated by the LacI protein via IPTG
induction. A large number of suitable vectors are known to those of
skill in the art, and are commercially available. The following
bacterial vectors are provided by way of example: pET (Novagen),
pET28, pBAD, pTrcHIS, pBR322, pQE70, pQE60, pQE-9 (Qiagen),
phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A,
pNH46A (Stratagene), ptrc99a, pKK223-3, pKK243-3, pDR540, pBR322,
pPS10, RSF1010, pRIT5 (Pharmacia); pCR (Invitrogen); pLex
(Invitrogen), and pUC plasmid derivatives.
[0129] A suitable expression vector contains regulatory sequences
that can be operably joined to an inserted nucleotide sequence
encoding the multivalent oligopeptide, DT, and/or PSM as described
herein. As used herein, the term "regulatory sequences" means
nucleotide sequences which are necessary for or conducive to the
transcription of an inserted sequence encoding a multivalent
oligopeptide, DT, and/or PSM as described herein by a host cell
and/or which are necessary for or conducive to the translation by a
host cell of the resulting transcript into the desired multivalent
oligopeptide, DT, and/or PSM. Regulatory sequences include, but are
not limited to, 5' sequences such as operators, promoters and
ribosome binding sequences, and 3' sequences such as
polyadenylation signals or transcription terminators. Regulatory
sequences can also include enhancer sequences or upstream activator
sequences.
[0130] Generally, bacterial vectors will include origins of
replication and selectable markers, e.g., the ampicillin,
tetracycline, kanamycin, resistance genes of E. coli, permitting
transformation of the host cell and a promoter derived from a
highly-expressed gene to direct transcription of a downstream
structural sequence. Suitable promoters include, but are not
limited to, the T7 promoter, lambda (k) promoter, T5 promoter, and
lac promoter, or promoters derived from operons encoding glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), acid phosphatase,
or heat shock proteins, or inducible promoters like cadmium (pcad),
and beta-lactamase (pbla).
[0131] Once an expression vector is selected, the polynucleotide as
described herein can be cloned downstream of the promoter, for
example, in a polylinker region. The vector is transformed into an
appropriate bacterial strain, and DNA is prepared using standard
techniques. The orientation and DNA sequence of the polynucleotide
as well as all other elements included in the vector, are confirmed
using restriction mapping, DNA sequence analysis, and/or PCR
analysis. Bacterial cells harboring the correct plasmid can be
stored as cell banks.
V. Immunogenic and Pharmaceutical Compositions
[0132] Further disclosed are compositions, e.g., immunogenic or
pharmaceutical compositions that contain an effective amount of the
multivalent oligopeptide, DT, and/or PSM as described herein, or a
polynucleotide encoding the polypeptide of the disclosure.
Compositions as described herein can further comprise additional
immunogenic components, e.g., as a multivalent vaccine, as well as
carriers, excipients or adjuvants.
[0133] Compositions as described herein can be formulated according
to known methods. Suitable preparation methods are described, for
example, in Remington's Pharmaceutical Sciences, 19th Edition, A.
R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), which is
incorporated herein by reference in its entirety. Composition can
be in a variety of forms, including, but not limited to an aqueous
solution, an emulsion, a gel, a suspension, lyophilized form, or
any other form known in the art. In addition, the composition can
contain pharmaceutically acceptable additives including, for
example, diluents, binders, stabilizers, and preservatives. Once
formulated, compositions of the disclosure can be administered
directly to the subject. The subjects to be treated can be animals;
in particular, human subjects can be treated.
[0134] Carriers that can be used with compositions of the
disclosure are well known in the art, and include, without
limitation, e.g., thyroglobulin, albumins such as human serum
albumin, tetanus toxoid, and polyamino acids such as poly L-lysine,
poly L-glutamic acid, influenza, hepatitis B virus core protein,
and the like. A variety of aqueous carriers can be used, e.g.,
water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid
and the like. Compositions can be sterilized by conventional, well
known sterilization techniques, or can be sterile filtered. A
resulting composition can be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a
sterile solution prior to administration. Compositions can contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamineoleate, etc.
[0135] Certain compositions as described herein further include one
or more adjuvants, a substance added to an immunogenic composition
to, for example, enhance, sustain, localize, or modulate an immune
response to an immunogen. The term "adjuvant" refers to any
material having the ability to (1) alter or increase the immune
response to a particular antigen or (2) increase or aid an effect
of a pharmacological agent. Any compound which can increase the
expression, antigenicity or immunogenicity of the polypeptide is a
potential adjuvant. The term "immunogenic carrier" as used herein
refers to a first moiety, e.g., a polypeptide or fragment, variant,
or derivative thereof which enhances the immunogenicity of a second
polypeptide or fragment, variant, or derivative thereof.
[0136] A great variety of materials have been shown to have
adjuvant activity through a variety of mechanisms. For example, an
increase in humoral immunity is typically manifested by a
significant increase in the titer of antibodies raised to the
antigen, and an increase in T-cell activity is typically manifested
in increased cell proliferation, or cellular cytotoxicity, or
cytokine secretion. An adjuvant can also alter or modulate an
immune response, for example, by changing a primarily humoral or
Th.sub.2 response into a primarily cellular, or Th.sub.1 response.
Immune responses to a given antigen can be tested by various
immunoassays well known to those of ordinary skill in the art,
and/or described elsewhere herein.
[0137] A wide number of adjuvants are familiar to persons of
ordinary skill in the art, and are described in numerous
references. Adjuvants which can be used in compositions described
herein include, but are not limited to: inert carriers, such as
alum, bentonite, latex, and acrylic particles; incomplete Freund's
adjuvant, complete Freund's adjuvant; aluminum-based salts such as
aluminum hydroxide; Alhydrogel (Al(OH.sub.3)); aluminum phosphate
(AlPO.sub.4); calcium-based salts; silica; any TLR biological
ligand(s); IDC-1001 (also known as GLA-SE; glucopyranosyl lipid
adjuvant stable emulsion) (Coler et al., PLoS One, 2010. 5(10): p.
e13677; Coler et al., PLoS One, 2011. 6(1): p. e16333); CpG (Mullen
et al., PLoS One, 2008. 3(8): p. e2940), or any combination
thereof. The amount of adjuvant, how it is formulated, and how it
is administered all parameters which are well within the purview of
a person of ordinary skill in the art.
[0138] In some embodiments, a composition of the disclosure further
comprises a liposome or other particulate carrier, which can serve,
e.g., to stabilize a formulation, to target the formulation to a
particular tissue, such as lymphoid tissue, or to increase the
half-life of the polypeptide composition. Such particulate carriers
include emulsions, foams, micelles, insoluble monolayers, liquid
crystals, phospholipid dispersions, lamellar layers, iscoms, and
the like. In these preparations, the polypeptide described herein
can be incorporated as part of a liposome or other particle, or can
be delivered in conjunction with a liposome. Liposomes for use in
accordance with the disclosure can be formed from standard
vesicle-forming lipids, which generally include neutral and
negatively charged phospholipids and a sterol, such as cholesterol.
A composition comprising a liposome or other particulate suspension
as well as the polypeptide as described herein can be administered
intravenously, locally, topically, etc. in a dose which varies
according to, inter alia, the manner of administration, the
polypeptide being delivered, and the stage of the disease being
treated.
[0139] For solid compositions, conventional nontoxic solid carriers
can be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient, that is, the polypeptide as
described herein, often at a concentration of 25%-75%.
[0140] For aerosol or mucosal administration, the polypeptide as
described herein can be supplied in finely divided form, optionally
along with a surfactant and, propellant and/or a mucoadhesive,
e.g., chitosan. The surfactant must, of course, be pharmaceutically
acceptable, and in some embodiments soluble in the propellant.
Representative of such agents are the esters or partial esters of
fatty acids containing from 6 to 22 carbon atoms, such as caproic,
octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric
and oleic acids with an aliphatic polyhydric alcohol or its cyclic
anhydride. Mixed esters, such as mixed or natural glycerides can be
employed. The surfactant can constitute 0.1%-20% by weight of the
composition, in some embodiments 0.25-5% by weight. The balance of
the composition is ordinarily propellant, although an atomizer can
be used in which no propellant is necessary and other percentages
are adjusted accordingly. In some embodiments, the immunogenic
polypeptides can be incorporated within an aerodynamically light
particle, such as those particles described in U.S. Pat. No.
6,942,868 or U.S. Pat. Pub. No. 2005/0008633. A carrier can also be
included, e.g., lecithin for intranasal delivery.
[0141] The disclosure is also directed to a method of producing the
composition according to the disclosure. In some embodiments, the
method of producing the composition comprises (a) isolating a
polypeptide according to the disclosure; and (b) adding an
adjuvant, carrier and/or excipient to the isolated polypeptide.
Some embodiments disclose further combining the polypeptide with
other staphylococcal antigens.
[0142] Some embodiments include a multivalent vaccine. A
multivalent vaccine of the present disclosure can include a
multivalent oligopeptide, DT, and/or PSM as described herein, or a
polynucleotide encoding a multivalent oligopeptide, DT, and/or PSM,
and one or more additional immunogenic components. Such components
can be additional immunogens of the same infectious agent, e.g., S.
aureus, or from other staphylococci, or can be immunogens derived
from other infectious agents which can be effectively,
conveniently, or economically administered together. In certain
embodiments, the multivalent oligopeptide, DT, and/or PSM as
described herein, can be combined with other toxins or other
virulent component-based vaccines to make a broad toxin-based
multivalent vaccine capable of targeting multiple bacterial
virulence determinants. In other embodiments, the multivalent
oligopeptide, DT, and/or PSM as described herein can be fused to
other immunogenic, biologically significant, or protective epitope
containing polypeptides to generate a multivalent vaccine in a
single chain and induce an immune response against multiple
antigens. In yet another embodiment, the multivalent oligopeptide,
DT, and/or PSM as described herein, can be fused to one or more T
cell epitopes to induce T cell immunity.
VI. Methods of Treatment/Prevention and Regimens
[0143] Also provided is a method of treating or preventing
Staphylococcus infection, e.g., S. aureus infection or treating or
preventing a disease caused by Staphylococcus, e.g, S. aureus in a
subject, comprising administering to a subject in need thereof a
composition as described herein comprising a multivalent
oligopeptide, DT, and/or PSM as described herein, or
polynucleotides, vectors, or host cells encoding same. In certain
embodiments, the subject is an animal, e.g., a vertebrate, e.g., a
mammal, e.g., a human. Some embodiments include a method of
inducing an immune response against a S. aureus strain, comprising
administering to a subject in need of said immune response an
effective amount of a composition comprising a multivalent
oligopeptide, DT, and/or PSM as described herein, or
polynucleotides, vectors, or host cells encoding same.
[0144] In some embodiments, a subject is administered a composition
comprising a multivalent oligopeptide, DT, and/or PSM as described
herein, or polynucleotides, vectors, or host cells encoding same
prophylactically, e.g., as a prophylactic vaccine, to establish or
enhance immunity to Staphylococcus, e.g., S. aureus, in a healthy
animal prior to potential or actual exposure to Staphylococcus,
e.g., S. aureus or contraction of a Staphylococcus-related symptom,
thus preventing disease, alleviating symptoms, reducing symptoms,
or reducing the severity of disease symptoms. In one embodiment the
disease is a respiratory disease, e.g., pneumonia. Other diseases
or conditions to be treated or prevented include, but are not
limited to, bacteremia, sepsis, skin infections, wound infections,
endocarditis, bone and joint infections, osteomyelitis, and/or
meningitis. One or more compositions, polypeptides,
polynucleotides, vectors, or host cells as described herein can
also be used to treat a subject already exposed to Staphylococcus,
e.g., S. aureus, or already suffering from a Staphylococcus related
symptom to further stimulate the immune system of the animal, thus
reducing or eliminating the symptoms associated with that exposure.
As defined herein, "treatment of an animal" refers to the use of
one or more compositions, polypeptides, polynucleotides, vectors,
or host cells of the disclosure to prevent, cure, retard, or reduce
the severity of S. aureus symptoms in an animal, and/or result in
no worsening of S. aureus symptoms over a specified period of time.
It is not required that any composition, polypeptide,
polynucleotide, a vector, or a host cell as described herein
provides total protection against a staphylococcal infection or
totally cure or eliminate all Staphylococcus related symptoms.
[0145] As used herein, "a subject in need of therapeutic and/or
preventative immunity" refers to a subject in which it is desirable
to treat, i.e., to prevent, cure, retard, or reduce the severity of
Staphylococcus related symptoms, or result in no worsening of
Staphylococcus related symptoms over a specified period of time. As
used herein, "a subject in need of the immune response" refers to a
subject for which an immune response(s) against a S Staphylococcus
related disease is desired.
[0146] Treatment with pharmaceutical compositions comprising an
immunogenic composition, polypeptide or polynucleotide as described
herein can occur separately or in conjunction with other
treatments, as appropriate.
[0147] In therapeutic applications, a composition, polypeptide or
polynucleotide of the disclosure is administered to a patient in an
amount sufficient to elicit an effective innate, humoral and/or
cellular response to the multivalent oligopeptide, DT, and/or PSM
to cure or at least partially arrest symptoms or complications.
[0148] An amount adequate to accomplish this is defined as
"therapeutically effective dose" or "unit dose." Amounts effective
for this use will depend on, e.g., the polypeptide or
polynucleotide composition, the manner of administration, the stage
and severity of the disease being treated, the weight and general
state of health of the patient, and the judgment of the prescribing
physician. In some embodiments, a priming dose is followed by a
boosting dose over a period of time.
[0149] In alternative embodiments, generally for humans an initial
immunization (that is for therapeutic or prophylactic
administration) is administered followed by boosting dosages in the
same dose range pursuant to a boosting regimen over weeks to months
depending upon the patient's response and condition by measuring
the antibody or T lymphocyte response in the patient's blood.
[0150] Polypeptides and compositions as described herein can
generally be employed in serious disease states, that is,
life-threatening or potentially life threatening situations. In
such cases, in view of the minimization of extraneous substances
and the relative nontoxic nature of the polypeptides, it is
possible and can be felt desirable by the treating physician to
administer substantial excesses of these polypeptide
compositions.
[0151] For therapeutic use, administration can begin at the first
sign of S. aureus infection or risk factors. In certain
embodiments, the initial dose is followed by boosting doses until,
e.g., symptoms are substantially abated and for a period
thereafter. In frequent infection, loading doses followed by
boosting doses can be required.
[0152] In certain embodiments, the composition as described herein
is delivered to a subject by methods described herein, thereby
achieving an effective immune response, and/or an effective
therapeutic or preventative immune response. Any mode of
administration can be used so long as the mode results in the
delivery and/or expression of the desired polypeptide in the
desired tissue, in an amount sufficient to generate an immune
response to Staphylococcus, e.g., S. aureus, and/or to generate a
prophylactically or therapeutically effective immune response to
Staphylococcus, e.g., to S. aureus, in an animal in need of such
response. According to the disclosed methods, a composition
described herein can be administered by mucosal delivery,
transdermal delivery, subcutaneous injection, intravenous
injection, oral administration, pulmonary administration,
intramuscular (i.m.) administration, or via intraperitoneal
injection. Other suitable routes of administration include, but not
limited to intratracheal, transdermal, intraocular, intranasal,
inhalation, intracavity, intraductal (e.g., into the pancreas) and
intraparenchymal (i.e., into any tissue) administration.
Transdermal delivery includes, but not limited to intradermal
(e.g., into the dermis or epidermis), transdermal (e.g.,
percutaneous) and transmucosal administration (i.e., into or
through skin or mucosal tissue). Intracavity administration
includes, but not limited to administration into oral, vaginal,
rectal, nasal, peritoneal, or intestinal cavities as well as,
intrathecal (i.e., into spinal canal), intraventricular (i.e., into
the brain ventricles or the heart ventricles), intra-arterial
(i.e., into the heart atrium) and sub arachnoidal (i.e., into the
sub arachnoid spaces of the brain) administration.
[0153] Any mode of administration can be used so long as the mode
results in the delivery and/or expression of the desired
polypeptide in an amount sufficient to generate an immune response
to Staphylococcus, e.g., S. aureus, and/or to generate a
prophylactically or therapeutically effective immune response to
Staphylococcus, e.g., S. aureus, in an animal in need of such
response. Administration as described herein can be by e.g., needle
injection, or other delivery or devices known in the art.
[0154] In some embodiments, a composition comprising a multivalent
oligopeptide, DT, and/or PSM as described herein, or
polynucleotides, vectors, or host cells encoding same, stimulate an
antibody response or a cell-mediated immune response sufficient for
protection of an animal against Staphylococcus, e.g., S. aureus
infection. In other embodiments, a composition comprising a
multivalent oligopeptide, DT, and/or PSM as described herein, or
polynucleotides, vectors, or host cells encoding same, stimulate
both a humoral and a cell-mediated response, the combination of
which is sufficient for protection of an animal against
Staphylococcus, e.g., S. aureus infection. In some embodiments, a
composition comprising a multivalent oligopeptide, DT, and/or PSM
as described herein, or polynucleotides, vectors, or host cells
encoding same, further stimulates an innate, an antibody, and/or a
cellular immune response.
[0155] In some embodiments, a composition comprising a multivalent
oligopeptide, DT, and/or PSM as described herein, or
polynucleotides, vectors, or host cells encoding same, can induce
antibody responses to S. aureus. In certain embodiments, components
that induce T cell responses (e.g., T cell epitopes) are combined
with components such as the polypeptides as described herein that
primarily induce an antibody response.
[0156] Further disclosed is a method for generating, enhancing, or
modulating a protective and/or therapeutic immune response to S.
aureus infection in a subject, comprising administering to a
subject in need of therapeutic and/or preventative immunity one or
more of the compositions as described herein.
[0157] The compositions as described herein can be administered to
an animal at any time during the lifecycle of the animal to which
it is being administered. In humans, administration of the
composition as described herein can, and often advantageously
occurs while other vaccines are being administered, e.g., as a
multivalent vaccine as described elsewhere herein.
[0158] Furthermore, the composition as described herein can be used
in any desired immunization or administration regimen; e.g., in a
single administration or alternatively as part of periodic
vaccination regimes such as annual vaccinations, or as in a
prime-boost regime in which composition or polypeptide or
polynucleotide of the disclosure is administered either before or
after the administration of the same or of a different polypeptide
or polynucleotide. Recent studies have indicated that a prime-boost
protocol is often a suitable method of administering vaccines. In a
prime-boost protocol, one or more compositions as described herein
can be utilized in a "prime boost" regimen. An example of a "prime
boost" regimen can be found in Yang, Z. et al. J. Virol.
77:799-803, 2002, which is incorporated herein by reference in its
entirety.
[0159] Infections to be treated include, but are not limited to a
localized or systemic infection of skin, soft tissue, blood, or an
organ or an auto-immune disease. Specific diseases or conditions to
be treated or prevented include, but are not limited to,
respiratory diseases, e.g., pneumonia, sepsis, skin infections,
wound infections, endocarditis, bone and joint infections,
osteomyelitis, and/or meningitis.
[0160] A number of animal models for S. aureus infection are known
in the art, and can be used with the methods disclosed herein
without undue experimentation. For example, a hamster model of
methicillin-resistant Staphylococcus aureus (MRSA) pneumonia has
been described for the testing of antimicrobials. (Verghese A. et
al., Chemotherapy. 34:497-503 (1988), Kephart P A. et al. J
Antimicrob Chemother. 21:33-9, (1988)). Further, a model of S.
aureus-induced pneumonia in adult, immunocompetent C57BL/6J mice is
described, which closely mimics the clinical and pathological
features of pneumonia in human patients. (Bubeck-Wardenburg J. et
al., Infect Immun. 75:1040-4 (2007)). Additionally, virulence has
been tested in a rat model of S. aureus pneumonia as described in
McElroy et al. (McElroy M C. et al., Infect Immun. 67:5541-4
(1999)). Finally, a standardized and reproducible model of
MRSA-induced septic pneumonia to evaluate new therapies was
established in sheep. (Enkhbaatar P. et al., Shock. 29(5):642-9
(2008)).
[0161] The practice of the disclosure will employ, unless otherwise
indicated, conventional techniques of cell biology, cell culture,
molecular biology, transgenic biology, microbiology, recombinant
DNA, and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature. See, for example,
Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al.,
ed., Cold Spring Harbor Laboratory Press: (1989); Molecular
Cloning: A Laboratory Manual, Sambrook et al., ed., Cold Springs
Harbor Laboratory, New York (1992), DNA Cloning, D. N. Glover ed.,
Volumes I and II (1985); Oligonucleotide Synthesis, M. J. Gait ed.,
(1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization, B. D. Hames & S. J. Higgins eds. (1984);
Transcription And Translation, B. D. Hames & S. J. Higgins eds.
(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss,
Inc., (1987); Immobilized Cells And Enzymes, IRL Press, (1986); B.
Perbal, A Practical Guide To Molecular Cloning (1984); the
treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene
Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos
eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology,
Vols. 154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell
And Molecular Biology, Mayer and Walker, eds., Academic Press,
London (1987); Handbook Of Experimental Immunology, Volumes I-IV,
D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the
Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989).
[0162] Standard reference works setting forth general principles of
immunology include Current Protocols in Immunology, John Wiley
& Sons, New York; Klein, J., Immunology: The Science of
Self-Nonself Discrimination, John Wiley & Sons, New York
(1982); Roitt, I., Brostoff, J. and Male D., Immunology, 6.sup.th
ed. London: Mosby (2001); Abbas A., Abul, A. and Lichtman, A.,
Cellular and Molecular Immunology, Ed. 5, Elsevier Health Sciences
Division (2005); and Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Press (1988).
EXAMPLES
[0163] The breadth and scope of the present disclosure should not
be limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims and
their equivalents.
Example 1: Generation of Attenuated Mutants of .delta.-Toxin and
PSM.alpha.3
[0164] The .delta.-toxin and PSM oligopeptides require an
amphiphilic .alpha.-helical structure to exhibit the surfactant
properties (Omae, et al., 2012, J Biol Chem, 287 (19):15570-15579;
Wang, et al., 2007, Nat Med, 13 (12):1510-1514). These peptides
consist of a hydrophobic and a hydrophilic surface as shown in FIG.
1A for the four PSM-.alpha. oligopeptides, two PSM-.beta.
oligopeptides, and the .delta.-toxin oligopeptide. These peptides
consist of a hydrophobic and a hydrophilic surface as shown in FIG.
1A for the four PSM-.alpha. oligopeptides, two PSM-.beta.
oligopeptides, and the .delta.-toxin oligopeptide. The hydrophobic
face of PSM-mec is shown in FIG. 1A (hydrophobic face shown in the
lower half of the circular depictions of the peptides). The
peptides are shown in Table 3, with hydrophobic face residues
underlined.
TABLE-US-00011 TABLE 3 OLIGOPEPTIDE SEQUENCES Sequence SEQ Peptide
(hydrophobic face residues underlined) ID NO .delta. toxin
MAQDIISTIGDLVKWIIDTVNKFTKK 1 PSM.alpha.1 MGIIAGIIKVIKSLIEQFTGK 38
PSM.alpha.2 MGIIAGIIKFIKGLIEKFTGK 12 PSM.alpha.3
MEFVAKLFKFFKDLLGKFLGNN 6 or 13 PSM.alpha.4 MAIVGTIIKIIKAIIDIFAK 14
PSM.beta.1 MEGLFNAIKDTVTAAINNDGAKLGTSIVNIVENGVGL 15 LSKLFGF
PSM.beta.2 MTGLAEAIANTVQAAQQHDSVKLGTSIVDIVANGVGL 16 LGKLFGF PSM-
MDFTGVITSIIDLIKTCIQAFG 52 mec
[0165] Helical wheel structures shown in FIG. 1 were created using
Heliquest software (heliquest.ipmc.cnrs.fr) to define and display
properties such as hydrophobicity and hydrophobic moment, net
charge (z) (Gautier, et al., 2008, Bioinformatics, 24
(18):2101-2102). Mutations disrupting the helical structure can
eliminate the surfactant properties (Omae, et al., 2012, J Biol
Chem, 287 (19):15570-15579) but could also decrease vaccine potency
of the mutant. Therefore, we generated mutants by minimizing the
disruption of the .alpha.-helix and to preserve immunogenicity of
peptides. To eliminate surfactant properties we replaced several
amino acids in the hydrophobic face of PSM.alpha.3 and
.delta.-toxin with less hydrophobic amino acids like alanine, and
glycine. The model predicts that these small amino acids will not
drastically change protein structure but will substantially
decrease hydrophobicity (FIG. 1B). We introduced mutations in two
residues in the hydrophobic face of both PSM.alpha.3 (V4 and L14)
and .delta.-toxin (L12 and V20). The mutants are shown in Table
4.
TABLE-US-00012 TABLE 4 MUTATED TOXIN SEQUENCES Delta toxin mutants
SEQUENCE (Mutated Residues Underlined) SEQ ID NO Wt
MAQDIISTIGDLVKWIIDTVNKFTKK 1 ALA-1 MAQDIISTIGDAVKWIIDTVNKFTKK 2
ALA-2 MAQDIISTIGDAVKWIIDTANKFTKK 3 GLY-1 MAQDIISTIGDGVKWIIDTVNKFTKK
4 GLY-2 MAQDIISTIGDGVKWIIDTGNKFTKK 5 PSM-.alpha.3 mutants WT
MEFVAKLFKFFKDLLGKFLGNN 6 or 13 ALA-1 MEFVAKLFKFFKDALGKFLGNN 7 ALA-2
MEFAAKLFKFFKDALGKFLGNN 8 GLY-1 MEFVAKLFKFFKDGLGKFLGNN 9 GLY-2
MEFGAKLFKFFKDGLGKFLGNN 10 GLY-ALA MEFGAKLFKFFKDALGKFLGNN 11
[0166] We engineered single and double mutants by replacing these
two amino acids either by alanine or by glycine or both. As shown
in FIG. 1B, the mutant constructs have decreased hydrophobicity and
hydrophobic moment compared to wild type toxin as analyzed by
Heliquest program (Gautier, et al., 2008, Bioinformatics, 24
(18):2101-2102). Similar mutants can be generated by further
mutations in the hydrophobic face as additional potential
attenuated toxoids.
Example 2: Evaluation of Attenuation of .delta.-Toxin and
PSM.alpha.3 Mutants
[0167] Mutations ala1, ala2, and gly2 were incorporated into delta
toxin sequence and the mutants were tested for lytic activity
towards horse red blood cells (HRBC) by the following method.
Toxicity assay using horse blood: 5 ml horse blood was centrifuged
at 2,000 RPM for 10 min at 20.degree. C. Supernatant was discarded
and pellet was washed once with 15 ml PBS. Required percentage of
the horse RBC cells was prepared in PBS by resuspending the pellet
(wt/vol). 100 .mu.l of the various oligopeptides were added in 100
.mu.l of horse RBC (different percentage as per required) in ELISA
dilution plate and then plates were incubated at 37.degree. C. for
45 min. Plates were then centrifuged and 100 .mu.l of the
supernatant was transferred into new NUNC ELISA plate. Absorbance
in the supernatant was determined in Versamax.TM. plate reader
(Molecular Devices CA) at 416 nm. The optical density of the
supernatant reflects the degree of hemolysis of red blood cells
caused by the toxin.
[0168] Neutralization Assay:
[0169] For neutralization, diluted serum samples were added to 50
.mu.l of the various oligopeptides (12.5 .mu.g/ml), incubated 10
min at room temperature, and then 100 .mu.l of 5% horse RBC were
added. The plates were incubated 37.degree. C. for 45 min. Plates
were then centrifuged and 100 .mu.l of the supernatant was
transferred into new NUNC ELISA plate. Absorbance in the
supernatants were determined in a Versamax.TM. plate reader
(Molecular Devices CA) at 416 nm.
[0170] As shown in FIG. 2, delta toxin mutants ala1 (L12A), ala2
(L12A/V20A), and gly2 (L12G/V20G) were fully attenuated. To test if
the mutants retained their neutralizing epitopes we tested the
delta toxin-ala2 mutant for binding to human neutralizing
antibodies in a pool of high titer human sera generated by
screening individual normal sera. As shown in FIG. 3, delta toxin
toxicity can be effectively neutralized with a 1:450 dilution of
human serum pool. Delta toxin-ala2 neither induced significant
lysis of the cells nor did it change the toxicity of delta toxin
when the toxin and mutant were combined. However, pre-incubation
with delta toxin-ala2 (22.5 .mu.g/ml) significantly reduced the
neutralizing capacity of the human sera (FIG. 3, compare 3.sup.rd
and 7.sup.th bar) towards delta toxin (11.25 .mu.g/ml). Since 2
fold excess of mutant can significantly reduce the neutralizing
capacity of the sera by .about.50% shows that the attenuated mutant
retains the ability to bind to neutralizing antibodies present in
the human serum.
[0171] We also generated mutants at positions V4 and L15 of
PSM.alpha.3. As shown in FIG. 4, mutation of both these sites in
PSM to glycine (PSM-gly2: V4A/L15A) strongly attenuated the toxin
while the other mutants showed partial attenuation.
Example 3: Generation of Fusion Constructs of AT62 with Delta Toxin
and PSM
[0172] The AT62 vaccine has shown efficacy when tested in different
animal models (Adhikari, et al., 2012, PLoS One, 7 (6):e38567). We
generated three fusion proteins with AT62 as the core subunit fused
to PSM.alpha.3, .delta.-toxin, or both using a flexible linker
(GGGGS; i.e. G4S) flanked with a C-terminal 6.times.His (FIGS.
5A&B). The nucleic acid sequences codon optimized for
expression in E. coli were prepared, and the three fusion proteins
were produced at small scale. The specificity and the size of the
constructs were confirmed by western blot with AT62 specific
antibodies (6C12) (FIG. 5C). The sequences are presented in Table
5. In each instance, the AT62 oligopeptide is single-underlined,
delta toxin is double-underlined, and PSM PSM.alpha.3 has a broken
underline. Additional fusion proteins including AT62 and
staphylococcal exotoxin B (SEB) are also presented
TABLE-US-00013 TABLE 5 AT62 FUSION OLIGOPEPTIDES Fusion SEQ ID
Codon-Optimized Nucleic SEQ ID Peptide Amino Acid Sequence NO Acid
Sequence NO AT62_DT ADSDINIKTGTTDIGSNTTVKT GDLVTYDKENGMHKKVFYSFI
DDKNHNKKLLVIRTKGTIAGG GGSGGGGSMAQDIISTIGDLVKWIIDTVNKFTKKHHHHHH 18
ATGGCGGATAGCGACATCAACATCA AAACGGGTACTACGGACATTGGCAG
CAATACGACCGTCAAGACCGGTGAT CTGGTCACCTATGACAAAGAGAATG
GTATGCACAAAAAGGTGTTTTACAG CTTCATTGATGACAAAAATCACAAC
AAGAAGCTGTTGGTTATTCGTACCA AAGGCACCATTGCCGGTGGTGGCGG
TTCCGGCGGTGGCGGTAGCATGGCA CAGGACATCATCTCTACCATCGGCG
ATCTGGTGAAATGGATCATTGATAC CGTTAACAAGTTCACGAAAAAGCAT
CATCACCATCACCACTGATAACTCG AGCACCACCACCACCACCACTGAGA TCCG 17
AT62_PSM ##STR00001## 20 ATGGCGGATAGCGACATCAACATCAA
AACGGGTACTACGGACATTGGCAGCA ATACGACCGTCAAGACCGGTGATCTG
GTCACCTATGACAAAGAGAATGGTAT GCACAAAAAGGTGTTTTACAGCTTCA
TTGATGACAAAAATCACAACAAGAAG CTGTTGGTTATTCGTACCAAAGGCACC
ATTGCCGGTGGTGGCGGCTCCGGTGG CGGTGGTTCTATGGAATTTGTTGCAAA
GCTGTTCAAATTCTTTAAGGATCTGCT GGGTAAATTCCTGGGCAACAACCATC
ATCACCATCACCACTGATAACT 19 AT62_DT_PSM ##STR00002## 22
ATGGCGGATAGCGACATCAACATCAA AACGGGTACTACGGACATTGGCAGCA
ATACGACCGTCAAGACCGGTGATCTG GTCACCTATGACAAAGAGAATGGTAT
GCACAAAAAGGTGTTTTACAGCTTCA TTGATGACAAAAATCACAACAAGAAG
CTGTTGGTTATTCGTACCAAAGGCACC ATTGCCGGTGGTGGTGGTTCTATGGCG
CAGGACATCATTTCCACGATCGGCGA TCTGGTTAAATGGATCATCGACACCGT
GAACAAGTTTACCAAGAAAGGTGGTG GCGGTAGCATGGAATTTGTTGCAAAA
CTGTTCAAATTCTTTAAGGATCTGCTG GGCAAGTTCCTGGGCAACAATCATCA
TCACCATCACCACTGATAA 21 AT62_rSEB MADSDINIKTGTTDIGSNTTVK
TGDLVTYDKENGMHKKVFYS FIDDKNHNKKLLVIRTKGTIAG GGGSGGGGSESQPDPKPDELH
KSSKFTGLMENMKVLYDDNH VSAINVKSIDQFRYFDLIYSIKD TKLGNYDNVRVEFKNKDLAD
KYKDKYVDVFGANAYYQCAF SKKTNDINSHQTDKRKTCMYG GVTEHNGNQLDKYRSITVRVF
EDGKNLLSFDVQTNKKKVTAQ ELDYLTRHYLVKNKKLYEFNN SPYETGYIKFIENENSFWYDM
MPAPGDKFDQSKYLMMYNDN KMVDSKDVKIEVYLTTKKK 23
CATATGGCAGACTCGGACATCAACAT CAAAACGGGCACGACGGACATTGGCT
CAAACACGACGGTGAAAACGGGCGAC CTGGTGACCTACGACAAAGAAAACGG
CATGCATAAAAAAGTGTTTTATAGCTT CATCGATGACAAAAACCACAACAAAA
AACTGCTGGTCATTCGTACCAAGGGT ACGATCGCAGGTGGTGGTGGTTCTGG
CGGTGGTGGTAGTGAATCCCAGCCGG ACCCGAAACCGGACGAACTGCATAAA
AGCTCTAAATTTACCGGCCTGATGGA AAATATGAAAGTGCTGTATGATGACA
ACCACGTGTCAGCCATTAATGTTAAAT CGATCGATCAATTCCGTTATTTCGACC
TGATTTACTCAATCAAAGATACCAAA CTGGGCAACTATGACAATGTGCGCGT
TGAATTCAAAAACAAAGATCTGGCAG ACAAATACAAAGATAAATACGTCGAC
GTGTTCGGTGCGAATGCCTATTACCAG TGCGCTTTCAGCAAGAAAACCAACGA
TATCAACTCTCATCAAACCGACAAAC GTAAAACGTGTATGTATGGCGGTGTG
ACCGAACACAACGGCAATCAGCTGGA TAAATACCGTAGTATCACGGTTCGCGT
CTTTGAAGATGGTAAAAACCTGCTGT CCTTCGATGTCCAGACCAACAAGAAA
AAAGTGACGGCACAAGAACTGGATTA TCTGACCCGCCATTACCTGGTTAAAAA
CAAAAAACTGTACGAATTCAACAACT CACCGTATGAAACGGGCTACATCAAA
TTCATCGAAAACGAAAACTCGTTCTG GTACGATATGATGCCGGCCCCGGGCG
ATAAATTCGACCAGTCCAAATATCTG ATGATGTACAATGATAACAAAATGGT
TGACTCCAAAGATGTGAAAATCGAAG TTTACCTGACGACGAAAAAAAAATAA GGATCC 25
rSEB_AT62 MESQPDPKPDELHKSSKFTGL MENMKVLYDDNHVSAINVKSI
DQFRYFDLIYSIKDTKLGNYDN VRVEFKNKDLADKYKDKYVD VFGANAYYQCAFSKKTNDINS
HQTDKRKTCMYGGVTEHNGN QLDKYRSITVRVFEDGKNLLSF DVQTNKKKVTAQELDYLTRH
YLVKNKKLYEFNNSPYETGYI KFIENENSFWYDMMPAPGDKF DQSKYLMMYNDNKMVDSKD
VKIEVYLTTKKKGGGGSGGGG SADSDFNIKTGTTDIGSNTTVKT GDLVTYDKENGMHKKVFYSFI
DDKNHNKKLLVIRTKGTIA 26 CATATGGAAAGCCAACCGGACCCGAA
ACCGGACGAACTGCATAAAAGCTCAA AATTCACGGGCCTGATGGAAAACATG
AAAGTGCTGTACGACGATAACCATGT CAGTGCAATTAATGTGAAATCCATCG
ATCAGTTTCGTTATTTCGACCTGATTT ACTCAATCAAAGATACCAAACTGGGC
AACTATGACAATGTGCGCGTTGAATT CAAAAACAAAGATCTGGCAGACAAAT
ACAAAGATAAATACGTCGACGTGTTC GGTGCGAATGCCTATTACCAGTGCGC
TTTCAGCAAGAAAACCAACGATATTA ATTCGCATCAAACCGACAAACGTAAA
ACGTGTATGTATGGCGGTGTCACCGA ACACAACGGCAATCAACTGGATAAAT
ACCGTAGCATCACGGTTCGCGTCTTTG AAGATGGTAAAAACCTGCTGTCTTTC
GACGTGCAGACCAACAAGAAAAAAGT TACGGCGCAAGAACTGGATTATCTGA
CCCGCCATTACCTGGTTAAAAACAAA AAACTGTACGAATTCAACAACTCACC
GTATGAAACGGGCTACATCAAATTCA TCGAAAACGAAAACTCGTTCTGGTAC
GATATGATGCCGGCCCCGGGCGATAA ATTCGACCAGAGTAAATACCTGATGA
TGTACAACGATAACAAAATGGTGGAT TCCAAAGACGTGAAAATTGAAGTTTA
TCTGACCACCAAGAAAAAAGGTGGTG GTGGTAGCGGTGGTGGTGGTAGCGCC
GATTCTGACATTAACATCAAAACCGG CACCACGGATATCGGTTCTAATACCA
CGGTTAAAACCGGCGATCTGGTCACG TATGACAAAGAAAACGGTATGCACAA
AAAAGTGTTTTATTCCTTCATTGACGA CAAAAATCACAACAAAAAACTGCTGG
TTATCCGCACGAAAGGCACCATCGCA TAAGGATCC 28 AT62_rSEB_DT
MADSDINIKTGTTDIGSNTTV KTGDLVTYDKENGMHKKVF YSFIDDKNHNKKLLVIRTKGT
IAGGGGSESQPDPKPDELHKS SKFTGLMENMKVLYDDNHV SAINVKSIDQFRYFDLIYSIKD
TKLGNYDNVRVEFKNKDLA DKYKDKYVDVFGANAYYQC AFSKKTNDINSHQTDKRKTC
MYGGVTEHNGNQLDKYRSIT VRVFEDGKNLLSFDVQTNKK KVTAQELDYLTRHYLVKNKK
LYEFNNSPYETGYIKFIENENS FWYDMMPAPGDKFDQSKYL MMYNDNKMVDSKDVKIEVY
LTTKKKGGGGSMAQDIISTIG DLVKWIIDTVNKFTKK. 29
CATATGGCAGATAGCGACATCAACAT CAAGACGGGCACGACGGACATTGGCT
CAAACACGACGGTGAAAACGGGTGAC CTGGTTACCTACGATAAAGAAAACGG
CATGCATAAGAAGGTGTTTTATTCTTT CATCGATGACAAAAACCACAATAAAA
AGCTGCTGGTTATTCGTACCAAGGGT ACGATTGCGGGCGGTGGCGGTAGTGA
ATCCCAGCCGGACCCGAAACCGGACG AACTGCATAAGAGCTCTAAATTTACC
GGCCTGATGGAAAATATGAAAGTGCT GTATGATGACAACCACGTCTCAGCCA
TTAATGTGAAATCGATCGATCAATTTC GTTATTTCGACCTGATTTACAGCATCA
AGGATACCAAACTGGGCAACTACGAC AATGTGCGCGTTGAATTTAAAAACAA
GGATCTGGCAGACAAATATAAGGATA AATACGTCGACGTGTTTGGTGCGAAT
GCCTATTACCAGTGCGCTTTCAGTAAA AAGACCAACGATATCAACTCCCATCA
AACCGACAAGCGTAAAACGTGTATGT ATGGCGGTGTCACCGAACACAACGGC
AATCAGCTGGATAAATACCGTTCAAT CACGGTTCGCGTCTTTGAAGATGGTA
AAAACCTGCTGTCGTTCGATGTTCAGA CCAATAAAAAGAAAGTCACGGCACAA
GAACTGGATTATCTGACCCGCCATTAC CTGGTTAAGAACAAGAAGCTGTACGA
ATTCAACAACAGTCCGTATGAAACGG GCTACATCAAGTTCATCGAAAACGAA
AACAGCTTCTGGTACGATATGATGCC GGCACCGGGTGATAAGTTCGACCAGA
GCAAGTACCTGATGATGTACAACGAT AACAAGATGGTTGATTCTAAGGACGT
GAAAATCGAAGTTTATCTGACCACGA AGAAAAAGGGCGGTGGCGGTAGCATG
GCTCAAGATATTATCTCTACCATCGGT GACCTGGTGAAGTGGATTATTGACAC
GGTGAACAAGTTTACGAAGAAATGAG GATCC 31
Example 4: Immunogenicity of Fusion Constructs
[0173] The immunogenicity of two of the fusion constructs (AT62_DT
and AT62_PSM) along with control (AT62 AA) was tested in Swiss
Webster mice in groups of 4, 4 and 8 mice respectively. Mice were
immunized subcutaneously with the proteins (10 .mu.g) along with
adjuvant (Sigma Adj System; an MPL based adjuvant) (5 .mu.g) three
times at two week intervals (days 0, 14 & 28). After the third
immunization the mice were boosted with the respective
.delta.-toxin or PSM.alpha.3 peptide (10 .mu.g) and serum samples
collected from the mice were tested for binding to the antigen
using ELISA as described before (Adhikari, et al., 2012, PLoS One,
7 (6):e38567). Briefly, 96-well plates were coated with 100 ng/well
of full length alpha toxin (List Biological Laboratories, Campbell,
Calif.), PSM.alpha.3, or delta toxin overnight at 4.degree. C.
Plates were blocked with Starting Block buffer (Thermo Scientific)
for one hour at room temperature (RT). Serum samples were diluted
at 1:100 using starting block buffer as diluent. Plates were washed
three times and sample dilutions were applied at 100 .mu.l/well.
Plates were incubated for one hour at RT and washed three times
before applying the conjugate, goat anti-mouse IgG (H&L)-HRP
(Horse Radish Peroxidase) in starting block buffer. Plates were
incubated for one hour at RT, washed as described above and
incubated with TMB (3,3',5,5'-tetramethylbenzidine) to detect HRP
for 30 min. Optical density at 650 nm was measured using a
Versamax.TM. plate reader (Molecular Devices CA).
[0174] As shown in FIG. 6, mice vaccinated with the fusion
constructs AT62-PSM and AT62-DT showed strong antibody response to
alpha hemolysin showing that the AT62 retained its immunogenicity
in the context of fusion construct. Response to PSM.alpha.3 and
.delta.-toxin peptides was also detectable although at a lower
level. These data suggest that induction of an antibody response to
both components is possible.
[0175] The following additional constructs will be constructed and
tested: [0176] Fusion of a single PSM.alpha.3 or .delta.-toxin or
2, 3, 4, 5, or 6 tandem repeats of PSM.alpha.3 or .delta.-toxin
(wild type or any of the mutants) at the N- or C-terminus of
attenuated LukS-PV mutants as described in PCT/US12/67483. [0177]
Fusion of a single PSM.alpha.3 or .delta.-toxin or 2, 3, 4, 5, or 6
tandem repeats of PSM.alpha.3 or .delta.-toxin (wild type or any of
the mutants) at the N- or C-terminus of attenuated superantigen
vaccines SEB.sub.L45R/Y89A/Y94A, SEA.sub.L48R/D70R/Y92A, or
TSST-1.sub.L30R/D27A/I46A. [0178] Fusion of a single PSM.alpha.3 or
.delta.-toxin or 2, 3, 4, 5, or 6 tandem repeats of PSM.alpha.3 or
.delta.-toxin (wild type or any of the mutants) along with AT62 to
any of the superantigen vaccines SEB.sub.L45R/Y89A/Y94A,
SEA.sub.L48R/D70R/Y92A, or TSST-1.sub.L30R/D27A/I46A.
[0179] An example of three potential fusion constructs is
schematically shown in FIG. 7.
Example 5: Triple Fusion Mutant of Staphylococcal Superantigen
Toxoids
[0180] The most prevalent S. aureus Superantigens (Sags) are SEB,
SEC, SEA, and TSST-1. Recombinant vaccines for SEB, SEA, and TSST-1
(subject of U.S. Pat. Nos. 6,713,284; 6,399,332; 7,087,235;
7,750,132; 7,378,257, and 8,067,202) were developed and tested
individually for protective efficacy in models of toxic shock
syndrome (Bavari, et al., 1996, J Infect Dis; Bavari and Ulrich,
1995, Infect Immun, 63 (2):423-429; Boles, et al., 2003 Clin
Immunol, 108 (1):51-59; Boles, et al., 2003, Vaccine, 21
(21-22):2791-2796; Ulrich, et al., 1998, Vaccine, 16
(19):1857-1864). Whereas the SAgs play an important role in
complications of SA disease, a major obstacle in developing
vaccines based on SAgs is the fact that there are >20 variants
of these toxins in various SA strains.
[0181] We evaluated the ability of human antibodies to SEB, SEA,
and TSST-1 to neutralize a wide range of S. aureus Sags, by the
following methods.
[0182] Affinity Purification of Human Anti-SAg Antibodies.
[0183] SEA, SEB and TSST-1 were coupled to agarose beads (1 mg SAg
per 1 mL bead volume) of an Aminolink.RTM. plus immobilization
column (Thermo Scientific, Rockford, Ill.) following the
manufacturer's protocol. Affinity purification of specific
antibodies from IVIG (Omrix Biopharmaceuticals, Nes-Ziona, Israel)
was carried out according to manufacturer's protocol with minor
modifications: 50 mL of IVIG was incubated with toxin-coupled beads
for 1 h 30 min at RT with gentle rocking, centrifuged, the
supernatant removed and a fresh 50 mL of IVIG incubated with the
beads for another 1 h and 30 min. Elution was performed with
glycine HCl pH 2.5 buffer. To avoid degradation of proteins eluted
fractions were collected in neutralizing buffer, containing (0.1 M
Tris) pH 9 to give a final pH between 6-7. The concentration of the
affinity purified antibodies was determined by BCA assay.
[0184] Toxin Neutralization Assay In Vitro.
[0185] Peripheral blood mononuclear cells were isolated from
heparinized blood of de-identified healthy human donors by Ficoll
gradient centrifugation as described elsewhere (Berthold, 1981,
Blut, 43 (6):367-371). Isolated peripheral blood mononuclear cells
were washed twice in PBS, frozen in 10% DMSO in heat-inactivated
fetal bovine serum (HI-FBS) overnight at -80.degree. C., and stored
in liquid nitrogen until further use. For the assay, cells were
washed and re-suspended in RPMI 1640 medium, supplemented with 5%
fetal bovine serum (FBS), non-essential amino acids,
Penicillin/Streptomycin and L-Glutamine. Cells were, enumerated by
Trypan blue exclusion and adjusted to 2.times.10.sup.6 cells/ml. 75
.mu.l of this cell suspension (1.5.times.10.sup.5 cells) with a
viability of >95% was added the wells of a 96-well plate
containing antibody/antigen mixes in duplicates as follows: 37.5
.mu.l of affinity-purified anti-SEA, -SEB, -TSST-1, in semi-log
dilutions (0.02-20 .mu.g/ml) or IVIG in semi log dilutions
(2.5-2500 .mu.g/ml) IVIG and 37.5 .mu.l of a 1 ng/ml preparation of
either SEB, SEC1-3, SEE, SEH, SEI, SEK, TSST-1, SpeC, or 2 ng/ml of
SED, or 3 ng/ml of SpeA. To test the synergistic activity of
purified polyclonal Abs a combination of anti-SEA, -SEB, and
-TSST-1 was used in a semi log dilution ranging from 0.02 to 20
.mu.g/ml and the same amount of toxin as above. Wells containing
medium with toxin only were served as positive controls. The plates
were incubated at 37.degree. C. in an atmosphere of 5% CO.sub.2-95%
air for 48 hours. Plates were centrifuged at 1600.times.g for 10
minutes, culture supernatants harvested and IFN.gamma.
concentration (pg/ml), was determined by ELISA (Human IFN-gamma
DuoSet, R&D Systems, Minneapolis, Minn.) following the
manufacturers' protocol. Plates were read at 450 nm using the
Versamax plate reader and data was transferred to and analyzed in
Excel. Positive control wells were considered to have a 0%
IFN.gamma. inhibition and, inhibition of IFN.gamma. production in
the presence of affinity purified antibodies was calculated as the
difference in IFN.gamma. concentration between the positive control
and sample. IC.sub.50 (the molar concentration of antibodies that
was required to reach 50% inhibition of IFN.gamma. production)
values for the neutralizing agents (purified antibodies or IVIG)
were determined using a 4-parameter logistic model (equation 205,
XLfit version 5.2).
[0186] As shown in FIG. 8, affinity purified human antibodies (from
IVIG) to each of these three toxins provided robust neutralization
towards the homologous toxin and varying degree of cross
neutralization to several other SAgs. However, a cocktail of the
three human antibodies resulted in a remarkable widening of the
cross neutralization activity.
[0187] In view of these results, novel fusions of the three toxoid
superantigens: SEB.sub.L45R/Y89A/Y94A, SEA.sub.L48R/D70R/Y92A, and
TSST-1.sub.L30R/D27A/I46A mutants are expressed as single molecules
in a prokaryotic host, e.g., E. coli. Such fusion proteins can be
superior to individual components not only due to ease of
manufacturing but also because they can enhance the elicitation of
cross reactive antibodies between various superantigens as the
common epitopes brought together into a single molecule can act in
an immunodominant manner. The following fusion proteins are to be
constructed.
[0188] A fusion of SEB.sub.L45R/Y89A/Y94A, SEA.sub.L48R/D70R/Y92A,
and TSST-1.sub.L30R/D27A/I46A mutants in one of the following
orders with a commonly used linker (L) such as but not limited to a
linker consisting of one or more repeats of four glycines and one
serine: [0189] BAT Fusion:
SEB.sub.L45R/Y89A/Y94A-L-SEA.sub.L48R/D70R/Y92A-L-TSST-1.sub.L30R-
/D27A/I46A [0190] BTA Fusion:
SEB.sub.L45R/Y89A/Y94A-L-TSST-1.sub.L30R/D27A/I46A-L-SEA.sub.L48R/D70R/Y9-
2A [0191] ABT Fusion:
SEA.sub.L48R/D70R/Y92A-L-SEB.sub.L45R/Y89A/Y94A-L-TSST-1.sub.L30R/D27A/I4-
6A [0192] ATB Fusion:
SEA.sub.L48R/D70R/Y92A-L-TSST-1.sub.L30R/D27A/I46A-L-SEB.sub.L45R/Y89A/Y9-
4A [0193] TAB Fusion:
TSST-1.sub.L30R/D27A/I46A-L-SEA.sub.L48R/D70R/Y92A-L-SEB.sub.L45R/Y89A/Y9-
4A [0194] TBA Fusion:
TSST-1.sub.L30R/D27A/I46A-L-SEB.sub.L45R/Y89A/Y94A-L-SEA.sub.L48R/D70R/Y9-
2A
[0195] Representative sequences are presented in Table 6
TABLE-US-00014 TABLE 6 SAG FUSION PROTEINS. SEQ ID NO BAT Fusion:
MESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDT 32
SEB.sub.L45R/Y89A/Y94A-
KLGNYDNVRVEFKNKDLADKYKDKYVDVFGANAYYQCAFSKKTNDINSHQTDKRKTC L-
MYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVK
SEA.sub.L48R/D70R/Y92A-
NKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKD L-TSST-
VKIEVYLTTKKKGGGSEKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKTENKE
1.sub.L30R/D27A/I46A
SHDQFRQHTILFKGFFTDHSWYNDLLVRFDSKDIVDKYKGKKVDLYGAYAGYQCAGG
TPNKTACMYGGVTLHDNNRLTEEKKVPINLWLDGKQNTVPLETVKTNKKNVTVQELD
LQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIY
RDNKTINSENMHIDIYLYTSGGGGSSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGS
MRIKNTDGSISLIAFPSPYYSPAFTKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVT
NTEKLPTPIELPLKVKVHGKDSPLKYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRS
SDKTGGYWKITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKTIEAEIN BTA
MESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDT 33
Fusion: KLGNYDNVRVEFKNKDLADKYKDKYVDVFGANAYYQCAFSKKTNDINSHQTDKRKTC
SEB.sub.L45R/Y89A/Y94A-
MYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVK L-TSST-
NKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKD
1.sub.L30R/D27A/I46A-L-
VKIEVYLTTKKKGGGSSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGSMRIKNTDGS
SEA.sub.L48R/D70R/Y92A
ISLIAFPSPYYSPAFTKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVTNTEKLPTPI
ELPLKVKVHGKDSPLKYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKTGGYWK
ITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKTIEAEINGGGGSEKSEEINEKDLRK
KSELQGTALGNLKQIYYYNEKAKTENKESHDQFRQHTILFKGFFTDHSWYNDLLVRF
DSKDIVDKYKGKKVDLYGAYAGYQCAGGTPNKTACMYGGVTLHDNNRLTEEKKVPIN
LWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIV
FHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTINSENMHIDIYLYTS ABT
MEKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKTENKESHDQFRQHTILFKGF 34
Fusion: FTDHSWYNDLLVRFDSKDIVDKYKGKKVDLYGAYAGYQCAGGTPNKTACMYGGVTLH
SEA.sub.L48R/D70R/Y92A-L-
DNNRLTEEKKVPINLWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYN
SEB.sub.L45R/Y89A/Y94A-L-
SDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTINSENMHIDI
TSST-1.sub.L30R/D27A/I46A
YLYTSGGGGSESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYF
DLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANAYYQCAFSKKTNDINS
HQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELD
YLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYN
DNKMVDSKDVKIEVYLTTKKKGGGSSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGS
MRIKNTDGSISLIAFPSPYYSPAFTKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVT
NTEKLPTPIELPLKVKVHGKDSPLKYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRS
SDKTGGYWKITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKTIEAEIN ATB
MEKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKTENKESHDQFRQHTILFKGF 35
Fusion: FTDHSWYNDLLVRFDSKDIVDKYKGKKVDLYGAYAGYQCAGGTPNKTACMYGGVTLH
SEA.sub.L48R/D70R/Y92A-
DNNRLTEEKKVPINLWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYN L-TSST-
SDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTINSENMHIDI
1.sub.L30R/D27A/I46A-L-
YLYTSGGGGSSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGSMRIKNTDGSISLIAF
SEB.sub.L45R/Y89A/Y94A
PSPYYSPAFTKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVTNTEKLPTPIELPLKV
KVHGKDSPLKYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKTGGYWKITMNDG
STYQSDLSKKFEYNTEKPPINIDEIKTIEAEINGGGSESQPDPKPDELHKSSKFTGL
MENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKY
KDKYVDVFGANAYYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITV
RVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIE
NENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK TAB Fusion: TSST-
MSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGSMRIKNTDGSISLIAFPSPYYSPAF 36
1.sub.L30R/D27A/I46A-L-
TKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVTNTEKLPTPIELPLKVKVHGKDSPL
SEA.sub.L48R/D70R/Y92A-L-
KYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKTGGYWKITMNDGSTYQSDLSK
SEB.sub.L45R/Y89A/Y94A
KFEYNTEKPPINIDEIKTIEAEINGGGSEKSEEINEKDLRKKSELQGTALGNLKQIY
YYNEKAKTENKESHDQFRQHTILFKGFFTDHSWYNDLLVRFDSKDIVDKYKGKKVDL
YGAYAGYQCAGGTPNKTACMYGGVTLHDNNRLTEEKKVPINLWLDGKQNTVPLETVK
TNKKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGA
QGQYSNTLLRIYRDNKTINSENMHIDIYLYTSGGGGSESQPDPKPDELHKSSKFTGL
MENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKY
KDKYVDVFGANAYYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITV
RVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIE
NENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK TBA Fusion: TSST-
MSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGSMRIKNTDGSISLIAFPSPYYSPAF 37
1.sub.L30R/D27A/I46A-L-
TKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVTNTEKLPTPIELPLKVKVHGKDSPL
SEB.sub.L45R/Y89A/Y94A-L-
KYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKTGGYWKITMNDGSTYQSDLSK
SEA.sub.L48R/D70R/Y92A
KFEYNTEKPPINIDEIKTIEAEINGGGGSESQPDPKPDELHKSSKFTGLMENMKVLY
DDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVF
GANAYYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKN
LLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYD
MMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKKGGGSEKSEEINEKDLRK
KSELQGTALGNLKQIYYYNEKAKTENKESHDQFRQHTILFKGFFTDHSWYNDLLVRF
DSKDIVDKYKGKKVDLYGAYAGYQCAGGTPNKTACMYGGVTLHDNNRLTEEKKVPIN
LWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIV
FHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTINSENMHIDIYLYTS
Sequence CWU 1
1
66126PRTStaphylococcus aureus 1Met Ala Gln Asp Ile Ile Ser Thr Ile
Gly Asp Leu Val Lys Trp Ile 1 5 10 15 Ile Asp Thr Val Asn Lys Phe
Thr Lys Lys 20 25 226PRTArtificialsynthetic 2Met Ala Gln Asp Ile
Ile Ser Thr Ile Gly Asp Ala Val Lys Trp Ile 1 5 10 15 Ile Asp Thr
Val Asn Lys Phe Thr Lys Lys 20 25 326PRTArtificialsynthetic 3Met
Ala Gln Asp Ile Ile Ser Thr Ile Gly Asp Ala Val Lys Trp Ile 1 5 10
15 Ile Asp Thr Ala Asn Lys Phe Thr Lys Lys 20 25
426PRTArtificialsynthetic 4Met Ala Gln Asp Ile Ile Ser Thr Ile Gly
Asp Gly Val Lys Trp Ile 1 5 10 15 Ile Asp Thr Val Asn Lys Phe Thr
Lys Lys 20 25 526PRTArtificialsynthetic 5Met Ala Gln Asp Ile Ile
Ser Thr Ile Gly Asp Gly Val Lys Trp Ile 1 5 10 15 Ile Asp Thr Gly
Asn Lys Phe Thr Lys Lys 20 25 622PRTStaphylococcus aureus 6Met Glu
Phe Val Ala Lys Leu Phe Lys Phe Phe Lys Asp Leu Leu Gly 1 5 10 15
Lys Phe Leu Gly Asn Asn 20 722PRTArtificialsynthetic 7Met Glu Phe
Val Ala Lys Leu Phe Lys Phe Phe Lys Asp Ala Leu Gly 1 5 10 15 Lys
Phe Leu Gly Asn Asn 20 822PRTArtificialsynthetic 8Met Glu Phe Ala
Ala Lys Leu Phe Lys Phe Phe Lys Asp Ala Leu Gly 1 5 10 15 Lys Phe
Leu Gly Asn Asn 20 922PRTArtificialsynthetic 9Met Glu Phe Val Ala
Lys Leu Phe Lys Phe Phe Lys Asp Gly Leu Gly 1 5 10 15 Lys Phe Leu
Gly Asn Asn 20 1022PRTArtificialsynthetic 10Met Glu Phe Gly Ala Lys
Leu Phe Lys Phe Phe Lys Asp Gly Leu Gly 1 5 10 15 Lys Phe Leu Gly
Asn Asn 20 1122PRTArtificialsynthetic 11Met Glu Phe Gly Ala Lys Leu
Phe Lys Phe Phe Lys Asp Ala Leu Gly 1 5 10 15 Lys Phe Leu Gly Asn
Asn 20 1221PRTStaphylococcus aureus 12Met Gly Ile Ile Ala Gly Ile
Ile Lys Phe Ile Lys Gly Leu Ile Glu 1 5 10 15 Lys Phe Thr Gly Lys
20 1322PRTStaphylococcus aureus 13Met Glu Phe Val Ala Lys Leu Phe
Lys Phe Phe Lys Asp Leu Leu Gly 1 5 10 15 Lys Phe Leu Gly Asn Asn
20 1420PRTStaphylococcus aureus 14Met Ala Ile Val Gly Thr Ile Ile
Lys Ile Ile Lys Ala Ile Ile Asp 1 5 10 15 Ile Phe Ala Lys 20
1544PRTStaphylococcus aureus 15Met Glu Gly Leu Phe Asn Ala Ile Lys
Asp Thr Val Thr Ala Ala Ile 1 5 10 15 Asn Asn Asp Gly Ala Lys Leu
Gly Thr Ser Ile Val Asn Ile Val Glu 20 25 30 Asn Gly Val Gly Leu
Leu Ser Lys Leu Phe Gly Phe 35 40 1644PRTStaphylococcus aureus
16Met Thr Gly Leu Ala Glu Ala Ile Ala Asn Thr Val Gln Ala Ala Gln 1
5 10 15 Gln His Asp Ser Val Lys Leu Gly Thr Ser Ile Val Asp Ile Val
Ala 20 25 30 Asn Gly Val Gly Leu Leu Gly Lys Leu Phe Gly Phe 35 40
17354DNAArtificialsynthetic 17atggcggata gcgacatcaa catcaaaacg
ggtactacgg acattggcag caatacgacc 60gtcaagaccg gtgatctggt cacctatgac
aaagagaatg gtatgcacaa aaaggtgttt 120tacagcttca ttgatgacaa
aaatcacaac aagaagctgt tggttattcg taccaaaggc 180accattgccg
gtggtggcgg ttccggcggt ggcggtagca tggcacagga catcatctct
240accatcggcg atctggtgaa atggatcatt gataccgtta acaagttcac
gaaaaagcat 300catcaccatc accactgata actcgagcac caccaccacc
accactgaga tccg 35418104PRTArtificialsynthetic 18Ala Asp Ser Asp
Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr
Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30
Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35
40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly
Gly 50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Gln Asp Ile
Ile Ser Thr 65 70 75 80 Ile Gly Asp Leu Val Lys Trp Ile Ile Asp Thr
Val Asn Lys Phe Thr 85 90 95 Lys Lys His His His His His His 100
19311DNAArtificialsynthetic 19atggcggata gcgacatcaa catcaaaacg
ggtactacgg acattggcag caatacgacc 60gtcaagaccg gtgatctggt cacctatgac
aaagagaatg gtatgcacaa aaaggtgttt 120tacagcttca ttgatgacaa
aaatcacaac aagaagctgt tggttattcg taccaaaggc 180accattgccg
gtggtggcgg ctccggtggc ggtggttcta tggaatttgt tgcaaagctg
240ttcaaattct ttaaggatct gctgggtaaa ttcctgggca acaaccatca
tcaccatcac 300cactgataac t 31120101PRTArtificialsynthetic 20Met Ala
Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly 1 5 10 15
Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu 20
25 30 Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys
Asn 35 40 45 His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr
Ile Ala Gly 50 55 60 Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Glu
Phe Val Ala Lys Leu 65 70 75 80 Phe Lys Phe Phe Lys Asp Leu Leu Gly
Lys Phe Leu Gly Asn Asn His 85 90 95 His His His His His 100
21387DNAArtificialsynthetic 21atggcggata gcgacatcaa catcaaaacg
ggtactacgg acattggcag caatacgacc 60gtcaagaccg gtgatctggt cacctatgac
aaagagaatg gtatgcacaa aaaggtgttt 120tacagcttca ttgatgacaa
aaatcacaac aagaagctgt tggttattcg taccaaaggc 180accattgccg
gtggtggtgg ttctatggcg caggacatca tttccacgat cggcgatctg
240gttaaatgga tcatcgacac cgtgaacaag tttaccaaga aaggtggtgg
cggtagcatg 300gaatttgttg caaaactgtt caaattcttt aaggatctgc
tgggcaagtt cctgggcaac 360aatcatcatc accatcacca ctgataa
38722127PRTArtificialsynthetic 22Met Ala Asp Ser Asp Ile Asn Ile
Lys Thr Gly Thr Thr Asp Ile Gly 1 5 10 15 Ser Asn Thr Thr Val Lys
Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu 20 25 30 Asn Gly Met His
Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn 35 40 45 His Asn
Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly 50 55 60
Gly Gly Gly Ser Met Ala Gln Asp Ile Ile Ser Thr Ile Gly Asp Leu 65
70 75 80 Val Lys Trp Ile Ile Asp Thr Val Asn Lys Phe Thr Lys Lys
Gly Gly 85 90 95 Gly Gly Ser Met Glu Phe Val Ala Lys Leu Phe Lys
Phe Phe Lys Asp 100 105 110 Leu Leu Gly Lys Phe Leu Gly Asn Asn His
His His His His His 115 120 125 23312PRTArtificialsynthetic 23Met
Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly 1 5 10
15 Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu
20 25 30 Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp
Lys Asn 35 40 45 His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly
Thr Ile Ala Gly 50 55 60 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Ser Gln Pro Asp Pro Lys 65 70 75 80 Pro Asp Glu Leu His Lys Ser Ser
Lys Phe Thr Gly Leu Met Glu Asn 85 90 95 Met Lys Val Leu Tyr Asp
Asp Asn His Val Ser Ala Ile Asn Val Lys 100 105 110 Ser Ile Asp Gln
Phe Arg Tyr Phe Asp Leu Ile Tyr Ser Ile Lys Asp 115 120 125 Thr Lys
Leu Gly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys Asn Lys 130 135 140
Asp Leu Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe Gly Ala 145
150 155 160 Asn Ala Tyr Tyr Gln Cys Ala Phe Ser Lys Lys Thr Asn Asp
Ile Asn 165 170 175 Ser His Gln Thr Asp Lys Arg Lys Thr Cys Met Tyr
Gly Gly Val Thr 180 185 190 Glu His Asn Gly Asn Gln Leu Asp Lys Tyr
Arg Ser Ile Thr Val Arg 195 200 205 Val Phe Glu Asp Gly Lys Asn Leu
Leu Ser Phe Asp Val Gln Thr Asn 210 215 220 Lys Lys Lys Val Thr Ala
Gln Glu Leu Asp Tyr Leu Thr Arg His Tyr 225 230 235 240 Leu Val Lys
Asn Lys Lys Leu Tyr Glu Phe Asn Asn Ser Pro Tyr Glu 245 250 255 Thr
Gly Tyr Ile Lys Phe Ile Glu Asn Glu Asn Ser Phe Trp Tyr Asp 260 265
270 Met Met Pro Ala Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr Leu Met
275 280 285 Met Tyr Asn Asp Asn Lys Met Val Asp Ser Lys Asp Val Lys
Ile Glu 290 295 300 Val Tyr Leu Thr Thr Lys Lys Lys 305 310
24948DNAArtificialsynthetic 24catatggcag attctgatat taatattaaa
accggtacta cagatattgg aagcaatact 60acagtaaaaa caggtgattt agtcacttat
gataaagaaa atggcatgca caaaaaagta 120ttttatagtt ttatcgatga
taaaaatcat aataaaaaac tgctagttat tagaacgaaa 180ggtaccattg
ctgggggagg ggggagcggg ggagggggga gcgagagtca accagatcct
240aaaccagatg agttgcacaa atcgagtaaa ttcactggtt tgatggaaaa
tatgaaagtt 300ttgtatgatg ataatcatgt atcagcaata aacgttaaat
ctatagatca gtttcgttac 360tttgacttaa tatattctat taaggacact
aagttaggga attatgataa tgttcgagtc 420gaatttaaaa acaaagattt
agctgataaa tacaaagata aatacgtaga tgtgtttgga 480gctaatgcgt
attatcaatg tgcgttttct aaaaaaacga atgatattaa ttcgcatcaa
540actgacaaac gaaaaacttg tatgtatggt ggtgtaactg agcataatgg
aaaccaatta 600gataaatata gaagtattac tgttcgggta tttgaagatg
gtaaaaattt attatctttt 660gacgtacaaa ctaataagaa aaaggtgact
gctcaagaat tagattacct aactcgtcac 720tatttggtga aaaataaaaa
actctatgaa tttaacaact cgccttatga aacgggatat 780attaaattta
tagaaaatga gaatagcttt tggtatgaca tgatgcctgc accaggagat
840aaatttgacc aatctaaata tttaatgatg tacaatgaca ataaaatggt
tgattctaaa 900gatgtgaaga ttgaagttta tcttacgaca aagaaaaagt gaggatcc
94825948DNAArtificialsynthetic 25catatggcag actcggacat caacatcaaa
acgggcacga cggacattgg ctcaaacacg 60acggtgaaaa cgggcgacct ggtgacctac
gacaaagaaa acggcatgca taaaaaagtg 120ttttatagct tcatcgatga
caaaaaccac aacaaaaaac tgctggtcat tcgtaccaag 180ggtacgatcg
caggtggtgg tggttctggc ggtggtggta gtgaatccca gccggacccg
240aaaccggacg aactgcataa aagctctaaa tttaccggcc tgatggaaaa
tatgaaagtg 300ctgtatgatg acaaccacgt gtcagccatt aatgttaaat
cgatcgatca attccgttat 360ttcgacctga tttactcaat caaagatacc
aaactgggca actatgacaa tgtgcgcgtt 420gaattcaaaa acaaagatct
ggcagacaaa tacaaagata aatacgtcga cgtgttcggt 480gcgaatgcct
attaccagtg cgctttcagc aagaaaacca acgatatcaa ctctcatcaa
540accgacaaac gtaaaacgtg tatgtatggc ggtgtgaccg aacacaacgg
caatcagctg 600gataaatacc gtagtatcac ggttcgcgtc tttgaagatg
gtaaaaacct gctgtccttc 660gatgtccaga ccaacaagaa aaaagtgacg
gcacaagaac tggattatct gacccgccat 720tacctggtta aaaacaaaaa
actgtacgaa ttcaacaact caccgtatga aacgggctac 780atcaaattca
tcgaaaacga aaactcgttc tggtacgata tgatgccggc cccgggcgat
840aaattcgacc agtccaaata tctgatgatg tacaatgata acaaaatggt
tgactccaaa 900gatgtgaaaa tcgaagttta cctgacgacg aaaaaaaaat aaggatcc
94826312PRTArtificialsynthetic 26Met Glu Ser Gln Pro Asp Pro Lys
Pro Asp Glu Leu His Lys Ser Ser 1 5 10 15 Lys Phe Thr Gly Leu Met
Glu Asn Met Lys Val Leu Tyr Asp Asp Asn 20 25 30 His Val Ser Ala
Ile Asn Val Lys Ser Ile Asp Gln Phe Arg Tyr Phe 35 40 45 Asp Leu
Ile Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn 50 55 60
Val Arg Val Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp 65
70 75 80 Lys Tyr Val Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln Cys
Ala Phe 85 90 95 Ser Lys Lys Thr Asn Asp Ile Asn Ser His Gln Thr
Asp Lys Arg Lys 100 105 110 Thr Cys Met Tyr Gly Gly Val Thr Glu His
Asn Gly Asn Gln Leu Asp 115 120 125 Lys Tyr Arg Ser Ile Thr Val Arg
Val Phe Glu Asp Gly Lys Asn Leu 130 135 140 Leu Ser Phe Asp Val Gln
Thr Asn Lys Lys Lys Val Thr Ala Gln Glu 145 150 155 160 Leu Asp Tyr
Leu Thr Arg His Tyr Leu Val Lys Asn Lys Lys Leu Tyr 165 170 175 Glu
Phe Asn Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu 180 185
190 Asn Glu Asn Ser Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys
195 200 205 Phe Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys
Met Val 210 215 220 Asp Ser Lys Asp Val Lys Ile Glu Val Tyr Leu Thr
Thr Lys Lys Lys 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Asp Ser Asp Ile Asn 245 250 255 Ile Lys Thr Gly Thr Thr Asp
Ile Gly Ser Asn Thr Thr Val Lys Thr 260 265 270 Gly Asp Leu Val Thr
Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val 275 280 285 Phe Tyr Ser
Phe Ile Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val 290 295 300 Ile
Arg Thr Lys Gly Thr Ile Ala 305 310 27948DNAArtificialsynthetic
27catatggaga gtcaaccaga tcctaaacca gatgagttgc acaaatcgag taaattcact
60ggtttgatgg aaaatatgaa agttttgtat gatgataatc atgtatcagc aataaacgtt
120aaatctatag atcagtttcg ttactttgac ttaatatatt ctattaagga
cactaagtta 180gggaattatg ataatgttcg agtcgaattt aaaaacaaag
atttagctga taaatacaaa 240gataaatacg tagatgtgtt tggagctaat
gcgtattatc aatgtgcgtt ttctaaaaaa 300acgaatgata ttaattcgca
tcaaactgac aaacgaaaaa cttgtatgta tggtggtgta 360actgagcata
atggaaacca attagataaa tatagaagta ttactgttcg ggtatttgaa
420gatggtaaaa atttattatc ttttgacgta caaactaata agaaaaaggt
gactgctcaa 480gaattagatt acctaactcg tcactatttg gtgaaaaata
aaaaactcta tgaatttaac 540aactcgcctt atgaaacggg atatattaaa
tttatagaaa atgagaatag cttttggtat 600gacatgatgc ctgcaccagg
agataaattt gaccaatcta aatatttaat gatgtacaat 660gacaataaaa
tggttgattc taaagatgtg aagattgaag tttatcttac gacaaagaaa
720aaggggggag gggggagcgg gggagggggg agcgcagatt ctgatattaa
tattaaaacc 780ggtactacag atattggaag caatactaca gtaaaaacag
gtgatttagt cacttatgat 840aaagaaaatg gcatgcacaa aaaagtattt
tatagtttta tcgatgataa aaatcataat 900aaaaaactgc tagttattag
aacgaaaggt accattgctt gaggatcc 94828948DNAArtificialsynthetic
28catatggaaa gccaaccgga cccgaaaccg gacgaactgc ataaaagctc aaaattcacg
60ggcctgatgg aaaacatgaa agtgctgtac gacgataacc atgtcagtgc aattaatgtg
120aaatccatcg atcagtttcg ttatttcgac ctgatttact caatcaaaga
taccaaactg 180ggcaactatg acaatgtgcg cgttgaattc aaaaacaaag
atctggcaga caaatacaaa 240gataaatacg tcgacgtgtt cggtgcgaat
gcctattacc agtgcgcttt cagcaagaaa 300accaacgata ttaattcgca
tcaaaccgac aaacgtaaaa cgtgtatgta tggcggtgtc 360accgaacaca
acggcaatca actggataaa taccgtagca tcacggttcg cgtctttgaa
420gatggtaaaa acctgctgtc tttcgacgtg cagaccaaca agaaaaaagt
tacggcgcaa 480gaactggatt atctgacccg ccattacctg gttaaaaaca
aaaaactgta cgaattcaac 540aactcaccgt atgaaacggg ctacatcaaa
ttcatcgaaa acgaaaactc gttctggtac 600gatatgatgc cggccccggg
cgataaattc gaccagagta aatacctgat gatgtacaac 660gataacaaaa
tggtggattc caaagacgtg aaaattgaag tttatctgac caccaagaaa
720aaaggtggtg gtggtagcgg tggtggtggt agcgccgatt ctgacattaa
catcaaaacc 780ggcaccacgg atatcggttc taataccacg gttaaaaccg
gcgatctggt cacgtatgac 840aaagaaaacg gtatgcacaa aaaagtgttt
tattccttca ttgacgacaa aaatcacaac 900aaaaaactgc tggttatccg
cacgaaaggc accatcgcat aaggatcc 94829338PRTArtificialsynthetic 29Met
Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly 1 5 10
15 Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu
20 25 30 Asn Gly Met His Lys Lys Val Phe Tyr Ser
Phe Ile Asp Asp Lys Asn 35 40 45 His Asn Lys Lys Leu Leu Val Ile
Arg Thr Lys Gly Thr Ile Ala Gly 50 55 60 Gly Gly Gly Ser Glu Ser
Gln Pro Asp Pro Lys Pro Asp Glu Leu His 65 70 75 80 Lys Ser Ser Lys
Phe Thr Gly Leu Met Glu Asn Met Lys Val Leu Tyr 85 90 95 Asp Asp
Asn His Val Ser Ala Ile Asn Val Lys Ser Ile Asp Gln Phe 100 105 110
Arg Tyr Phe Asp Leu Ile Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn 115
120 125 Tyr Asp Asn Val Arg Val Glu Phe Lys Asn Lys Asp Leu Ala Asp
Lys 130 135 140 Tyr Lys Asp Lys Tyr Val Asp Val Phe Gly Ala Asn Ala
Tyr Tyr Gln 145 150 155 160 Cys Ala Phe Ser Lys Lys Thr Asn Asp Ile
Asn Ser His Gln Thr Asp 165 170 175 Lys Arg Lys Thr Cys Met Tyr Gly
Gly Val Thr Glu His Asn Gly Asn 180 185 190 Gln Leu Asp Lys Tyr Arg
Ser Ile Thr Val Arg Val Phe Glu Asp Gly 195 200 205 Lys Asn Leu Leu
Ser Phe Asp Val Gln Thr Asn Lys Lys Lys Val Thr 210 215 220 Ala Gln
Glu Leu Asp Tyr Leu Thr Arg His Tyr Leu Val Lys Asn Lys 225 230 235
240 Lys Leu Tyr Glu Phe Asn Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys
245 250 255 Phe Ile Glu Asn Glu Asn Ser Phe Trp Tyr Asp Met Met Pro
Ala Pro 260 265 270 Gly Asp Lys Phe Asp Gln Ser Lys Tyr Leu Met Met
Tyr Asn Asp Asn 275 280 285 Lys Met Val Asp Ser Lys Asp Val Lys Ile
Glu Val Tyr Leu Thr Thr 290 295 300 Lys Lys Lys Gly Gly Gly Gly Ser
Met Ala Gln Asp Ile Ile Ser Thr 305 310 315 320 Ile Gly Asp Leu Val
Lys Trp Ile Ile Asp Thr Val Asn Lys Phe Thr 325 330 335 Lys Lys
301026DNAArtificialsynthetic 30catatggcag attctgatat taatattaaa
accggtacta cagatattgg aagcaatact 60acagtaaaaa caggtgattt agtcacttat
gataaagaaa atggcatgca caaaaaagta 120ttttatagtt ttatcgatga
taaaaatcat aataaaaaac tgctagttat tagaacgaaa 180ggtaccattg
ctgggggagg ggggagcgag agtcaaccag atcctaaacc agatgagttg
240cacaaatcga gtaaattcac tggtttgatg gaaaatatga aagttttgta
tgatgataat 300catgtatcag caataaacgt taaatctata gatcagtttc
gttactttga cttaatatat 360tctattaagg acactaagtt agggaattat
gataatgttc gagtcgaatt taaaaacaaa 420gatttagctg ataaatacaa
agataaatac gtagatgtgt ttggagctaa tgcgtattat 480caatgtgcgt
tttctaaaaa aacgaatgat attaattcgc atcaaactga caaacgaaaa
540acttgtatgt atggtggtgt aactgagcat aatggaaacc aattagataa
atatagaagt 600attactgttc gggtatttga agatggtaaa aatttattat
cttttgacgt acaaactaat 660aagaaaaagg tgactgctca agaattagat
tacctaactc gtcactattt ggtgaaaaat 720aaaaaactct atgaatttaa
caactcgcct tatgaaacgg gatatattaa atttatagaa 780aatgagaata
gcttttggta tgacatgatg cctgcaccag gagataaatt tgaccaatct
840aaatatttaa tgatgtacaa tgacaataaa atggttgatt ctaaagatgt
gaagattgaa 900gtttatctta cgacaaagaa aaagggggga ggggggagca
tggcacaaga tatcatttca 960acaatcggtg acttagtaaa atggattatc
gacacagtga acaaattcac taaaaaatga 1020ggatcc
1026311026DNAArtificialsynthetic 31catatggcag atagcgacat caacatcaag
acgggcacga cggacattgg ctcaaacacg 60acggtgaaaa cgggtgacct ggttacctac
gataaagaaa acggcatgca taagaaggtg 120ttttattctt tcatcgatga
caaaaaccac aataaaaagc tgctggttat tcgtaccaag 180ggtacgattg
cgggcggtgg cggtagtgaa tcccagccgg acccgaaacc ggacgaactg
240cataagagct ctaaatttac cggcctgatg gaaaatatga aagtgctgta
tgatgacaac 300cacgtctcag ccattaatgt gaaatcgatc gatcaatttc
gttatttcga cctgatttac 360agcatcaagg ataccaaact gggcaactac
gacaatgtgc gcgttgaatt taaaaacaag 420gatctggcag acaaatataa
ggataaatac gtcgacgtgt ttggtgcgaa tgcctattac 480cagtgcgctt
tcagtaaaaa gaccaacgat atcaactccc atcaaaccga caagcgtaaa
540acgtgtatgt atggcggtgt caccgaacac aacggcaatc agctggataa
ataccgttca 600atcacggttc gcgtctttga agatggtaaa aacctgctgt
cgttcgatgt tcagaccaat 660aaaaagaaag tcacggcaca agaactggat
tatctgaccc gccattacct ggttaagaac 720aagaagctgt acgaattcaa
caacagtccg tatgaaacgg gctacatcaa gttcatcgaa 780aacgaaaaca
gcttctggta cgatatgatg ccggcaccgg gtgataagtt cgaccagagc
840aagtacctga tgatgtacaa cgataacaag atggttgatt ctaaggacgt
gaaaatcgaa 900gtttatctga ccacgaagaa aaagggcggt ggcggtagca
tggctcaaga tattatctct 960accatcggtg acctggtgaa gtggattatt
gacacggtga acaagtttac gaagaaatga 1020ggatcc
102632675PRTArtificialsynthetic 32Met Glu Ser Gln Pro Asp Pro Lys
Pro Asp Glu Leu His Lys Ser Ser 1 5 10 15 Lys Phe Thr Gly Leu Met
Glu Asn Met Lys Val Leu Tyr Asp Asp Asn 20 25 30 His Val Ser Ala
Ile Asn Val Lys Ser Ile Asp Gln Phe Arg Tyr Phe 35 40 45 Asp Leu
Ile Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn 50 55 60
Val Arg Val Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp 65
70 75 80 Lys Tyr Val Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln Cys
Ala Phe 85 90 95 Ser Lys Lys Thr Asn Asp Ile Asn Ser His Gln Thr
Asp Lys Arg Lys 100 105 110 Thr Cys Met Tyr Gly Gly Val Thr Glu His
Asn Gly Asn Gln Leu Asp 115 120 125 Lys Tyr Arg Ser Ile Thr Val Arg
Val Phe Glu Asp Gly Lys Asn Leu 130 135 140 Leu Ser Phe Asp Val Gln
Thr Asn Lys Lys Lys Val Thr Ala Gln Glu 145 150 155 160 Leu Asp Tyr
Leu Thr Arg His Tyr Leu Val Lys Asn Lys Lys Leu Tyr 165 170 175 Glu
Phe Asn Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu 180 185
190 Asn Glu Asn Ser Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys
195 200 205 Phe Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys
Met Val 210 215 220 Asp Ser Lys Asp Val Lys Ile Glu Val Tyr Leu Thr
Thr Lys Lys Lys 225 230 235 240 Gly Gly Gly Ser Glu Lys Ser Glu Glu
Ile Asn Glu Lys Asp Leu Arg 245 250 255 Lys Lys Ser Glu Leu Gln Gly
Thr Ala Leu Gly Asn Leu Lys Gln Ile 260 265 270 Tyr Tyr Tyr Asn Glu
Lys Ala Lys Thr Glu Asn Lys Glu Ser His Asp 275 280 285 Gln Phe Arg
Gln His Thr Ile Leu Phe Lys Gly Phe Phe Thr Asp His 290 295 300 Ser
Trp Tyr Asn Asp Leu Leu Val Arg Phe Asp Ser Lys Asp Ile Val 305 310
315 320 Asp Lys Tyr Lys Gly Lys Lys Val Asp Leu Tyr Gly Ala Tyr Ala
Gly 325 330 335 Tyr Gln Cys Ala Gly Gly Thr Pro Asn Lys Thr Ala Cys
Met Tyr Gly 340 345 350 Gly Val Thr Leu His Asp Asn Asn Arg Leu Thr
Glu Glu Lys Lys Val 355 360 365 Pro Ile Asn Leu Trp Leu Asp Gly Lys
Gln Asn Thr Val Pro Leu Glu 370 375 380 Thr Val Lys Thr Asn Lys Lys
Asn Val Thr Val Gln Glu Leu Asp Leu 385 390 395 400 Gln Ala Arg Arg
Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr Asn Ser Asp 405 410 415 Val Phe
Asp Gly Lys Val Gln Arg Gly Leu Ile Val Phe His Thr Ser 420 425 430
Thr Glu Pro Ser Val Asn Tyr Asp Leu Phe Gly Ala Gln Gly Gln Tyr 435
440 445 Ser Asn Thr Leu Leu Arg Ile Tyr Arg Asp Asn Lys Thr Ile Asn
Ser 450 455 460 Glu Asn Met His Ile Asp Ile Tyr Leu Tyr Thr Ser Gly
Gly Gly Gly 465 470 475 480 Ser Ser Thr Asn Asp Asn Ile Lys Asp Leu
Leu Asp Trp Tyr Ser Ser 485 490 495 Gly Ser Asp Thr Phe Thr Asn Ser
Glu Val Leu Ala Asn Ser Arg Gly 500 505 510 Ser Met Arg Ile Lys Asn
Thr Asp Gly Ser Ile Ser Leu Ile Ala Phe 515 520 525 Pro Ser Pro Tyr
Tyr Ser Pro Ala Phe Thr Lys Gly Glu Lys Val Asp 530 535 540 Leu Asn
Thr Lys Arg Thr Lys Lys Ser Gln His Thr Ser Glu Gly Thr 545 550 555
560 Tyr Ile His Phe Gln Ile Ser Gly Val Thr Asn Thr Glu Lys Leu Pro
565 570 575 Thr Pro Ile Glu Leu Pro Leu Lys Val Lys Val His Gly Lys
Asp Ser 580 585 590 Pro Leu Lys Tyr Trp Pro Lys Phe Asp Lys Lys Gln
Leu Ala Ile Ser 595 600 605 Thr Leu Asp Phe Glu Ile Arg His Gln Leu
Thr Gln Ile His Gly Leu 610 615 620 Tyr Arg Ser Ser Asp Lys Thr Gly
Gly Tyr Trp Lys Ile Thr Met Asn 625 630 635 640 Asp Gly Ser Thr Tyr
Gln Ser Asp Leu Ser Lys Lys Phe Glu Tyr Asn 645 650 655 Thr Glu Lys
Pro Pro Ile Asn Ile Asp Glu Ile Lys Thr Ile Glu Ala 660 665 670 Glu
Ile Asn 675 33675PRTArtificialsynthetic 33Met Glu Ser Gln Pro Asp
Pro Lys Pro Asp Glu Leu His Lys Ser Ser 1 5 10 15 Lys Phe Thr Gly
Leu Met Glu Asn Met Lys Val Leu Tyr Asp Asp Asn 20 25 30 His Val
Ser Ala Ile Asn Val Lys Ser Ile Asp Gln Phe Arg Tyr Phe 35 40 45
Asp Leu Ile Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn 50
55 60 Val Arg Val Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys
Asp 65 70 75 80 Lys Tyr Val Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln
Cys Ala Phe 85 90 95 Ser Lys Lys Thr Asn Asp Ile Asn Ser His Gln
Thr Asp Lys Arg Lys 100 105 110 Thr Cys Met Tyr Gly Gly Val Thr Glu
His Asn Gly Asn Gln Leu Asp 115 120 125 Lys Tyr Arg Ser Ile Thr Val
Arg Val Phe Glu Asp Gly Lys Asn Leu 130 135 140 Leu Ser Phe Asp Val
Gln Thr Asn Lys Lys Lys Val Thr Ala Gln Glu 145 150 155 160 Leu Asp
Tyr Leu Thr Arg His Tyr Leu Val Lys Asn Lys Lys Leu Tyr 165 170 175
Glu Phe Asn Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu 180
185 190 Asn Glu Asn Ser Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp
Lys 195 200 205 Phe Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn
Lys Met Val 210 215 220 Asp Ser Lys Asp Val Lys Ile Glu Val Tyr Leu
Thr Thr Lys Lys Lys 225 230 235 240 Gly Gly Gly Ser Ser Thr Asn Asp
Asn Ile Lys Asp Leu Leu Asp Trp 245 250 255 Tyr Ser Ser Gly Ser Asp
Thr Phe Thr Asn Ser Glu Val Leu Ala Asn 260 265 270 Ser Arg Gly Ser
Met Arg Ile Lys Asn Thr Asp Gly Ser Ile Ser Leu 275 280 285 Ile Ala
Phe Pro Ser Pro Tyr Tyr Ser Pro Ala Phe Thr Lys Gly Glu 290 295 300
Lys Val Asp Leu Asn Thr Lys Arg Thr Lys Lys Ser Gln His Thr Ser 305
310 315 320 Glu Gly Thr Tyr Ile His Phe Gln Ile Ser Gly Val Thr Asn
Thr Glu 325 330 335 Lys Leu Pro Thr Pro Ile Glu Leu Pro Leu Lys Val
Lys Val His Gly 340 345 350 Lys Asp Ser Pro Leu Lys Tyr Trp Pro Lys
Phe Asp Lys Lys Gln Leu 355 360 365 Ala Ile Ser Thr Leu Asp Phe Glu
Ile Arg His Gln Leu Thr Gln Ile 370 375 380 His Gly Leu Tyr Arg Ser
Ser Asp Lys Thr Gly Gly Tyr Trp Lys Ile 385 390 395 400 Thr Met Asn
Asp Gly Ser Thr Tyr Gln Ser Asp Leu Ser Lys Lys Phe 405 410 415 Glu
Tyr Asn Thr Glu Lys Pro Pro Ile Asn Ile Asp Glu Ile Lys Thr 420 425
430 Ile Glu Ala Glu Ile Asn Gly Gly Gly Gly Ser Glu Lys Ser Glu Glu
435 440 445 Ile Asn Glu Lys Asp Leu Arg Lys Lys Ser Glu Leu Gln Gly
Thr Ala 450 455 460 Leu Gly Asn Leu Lys Gln Ile Tyr Tyr Tyr Asn Glu
Lys Ala Lys Thr 465 470 475 480 Glu Asn Lys Glu Ser His Asp Gln Phe
Arg Gln His Thr Ile Leu Phe 485 490 495 Lys Gly Phe Phe Thr Asp His
Ser Trp Tyr Asn Asp Leu Leu Val Arg 500 505 510 Phe Asp Ser Lys Asp
Ile Val Asp Lys Tyr Lys Gly Lys Lys Val Asp 515 520 525 Leu Tyr Gly
Ala Tyr Ala Gly Tyr Gln Cys Ala Gly Gly Thr Pro Asn 530 535 540 Lys
Thr Ala Cys Met Tyr Gly Gly Val Thr Leu His Asp Asn Asn Arg 545 550
555 560 Leu Thr Glu Glu Lys Lys Val Pro Ile Asn Leu Trp Leu Asp Gly
Lys 565 570 575 Gln Asn Thr Val Pro Leu Glu Thr Val Lys Thr Asn Lys
Lys Asn Val 580 585 590 Thr Val Gln Glu Leu Asp Leu Gln Ala Arg Arg
Tyr Leu Gln Glu Lys 595 600 605 Tyr Asn Leu Tyr Asn Ser Asp Val Phe
Asp Gly Lys Val Gln Arg Gly 610 615 620 Leu Ile Val Phe His Thr Ser
Thr Glu Pro Ser Val Asn Tyr Asp Leu 625 630 635 640 Phe Gly Ala Gln
Gly Gln Tyr Ser Asn Thr Leu Leu Arg Ile Tyr Arg 645 650 655 Asp Asn
Lys Thr Ile Asn Ser Glu Asn Met His Ile Asp Ile Tyr Leu 660 665 670
Tyr Thr Ser 675 34675PRTArtificialsynthetic 34Met Glu Lys Ser Glu
Glu Ile Asn Glu Lys Asp Leu Arg Lys Lys Ser 1 5 10 15 Glu Leu Gln
Gly Thr Ala Leu Gly Asn Leu Lys Gln Ile Tyr Tyr Tyr 20 25 30 Asn
Glu Lys Ala Lys Thr Glu Asn Lys Glu Ser His Asp Gln Phe Arg 35 40
45 Gln His Thr Ile Leu Phe Lys Gly Phe Phe Thr Asp His Ser Trp Tyr
50 55 60 Asn Asp Leu Leu Val Arg Phe Asp Ser Lys Asp Ile Val Asp
Lys Tyr 65 70 75 80 Lys Gly Lys Lys Val Asp Leu Tyr Gly Ala Tyr Ala
Gly Tyr Gln Cys 85 90 95 Ala Gly Gly Thr Pro Asn Lys Thr Ala Cys
Met Tyr Gly Gly Val Thr 100 105 110 Leu His Asp Asn Asn Arg Leu Thr
Glu Glu Lys Lys Val Pro Ile Asn 115 120 125 Leu Trp Leu Asp Gly Lys
Gln Asn Thr Val Pro Leu Glu Thr Val Lys 130 135 140 Thr Asn Lys Lys
Asn Val Thr Val Gln Glu Leu Asp Leu Gln Ala Arg 145 150 155 160 Arg
Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr Asn Ser Asp Val Phe Asp 165 170
175 Gly Lys Val Gln Arg Gly Leu Ile Val Phe His Thr Ser Thr Glu Pro
180 185 190 Ser Val Asn Tyr Asp Leu Phe Gly Ala Gln Gly Gln Tyr Ser
Asn Thr 195 200 205 Leu Leu Arg Ile Tyr Arg Asp Asn Lys Thr Ile Asn
Ser Glu Asn Met 210 215 220 His Ile Asp Ile Tyr Leu Tyr Thr Ser Gly
Gly Gly Gly Ser Glu Ser 225 230 235 240 Gln Pro Asp Pro Lys Pro Asp
Glu Leu His Lys Ser Ser Lys Phe Thr 245 250 255 Gly Leu Met Glu Asn
Met Lys Val Leu Tyr Asp Asp Asn His Val Ser 260 265 270 Ala Ile Asn
Val Lys Ser Ile Asp Gln Phe Arg Tyr Phe Asp Leu Ile 275 280 285 Tyr
Ser Ile Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn Val Arg Val 290
295
300 Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp Lys Tyr Val
305 310 315 320 Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln Cys Ala Phe
Ser Lys Lys 325 330 335 Thr Asn Asp Ile Asn Ser His Gln Thr Asp Lys
Arg Lys Thr Cys Met 340 345 350 Tyr Gly Gly Val Thr Glu His Asn Gly
Asn Gln Leu Asp Lys Tyr Arg 355 360 365 Ser Ile Thr Val Arg Val Phe
Glu Asp Gly Lys Asn Leu Leu Ser Phe 370 375 380 Asp Val Gln Thr Asn
Lys Lys Lys Val Thr Ala Gln Glu Leu Asp Tyr 385 390 395 400 Leu Thr
Arg His Tyr Leu Val Lys Asn Lys Lys Leu Tyr Glu Phe Asn 405 410 415
Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu Asn Glu Asn 420
425 430 Ser Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys Phe Asp
Gln 435 440 445 Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Met Val
Asp Ser Lys 450 455 460 Asp Val Lys Ile Glu Val Tyr Leu Thr Thr Lys
Lys Lys Gly Gly Gly 465 470 475 480 Ser Ser Thr Asn Asp Asn Ile Lys
Asp Leu Leu Asp Trp Tyr Ser Ser 485 490 495 Gly Ser Asp Thr Phe Thr
Asn Ser Glu Val Leu Ala Asn Ser Arg Gly 500 505 510 Ser Met Arg Ile
Lys Asn Thr Asp Gly Ser Ile Ser Leu Ile Ala Phe 515 520 525 Pro Ser
Pro Tyr Tyr Ser Pro Ala Phe Thr Lys Gly Glu Lys Val Asp 530 535 540
Leu Asn Thr Lys Arg Thr Lys Lys Ser Gln His Thr Ser Glu Gly Thr 545
550 555 560 Tyr Ile His Phe Gln Ile Ser Gly Val Thr Asn Thr Glu Lys
Leu Pro 565 570 575 Thr Pro Ile Glu Leu Pro Leu Lys Val Lys Val His
Gly Lys Asp Ser 580 585 590 Pro Leu Lys Tyr Trp Pro Lys Phe Asp Lys
Lys Gln Leu Ala Ile Ser 595 600 605 Thr Leu Asp Phe Glu Ile Arg His
Gln Leu Thr Gln Ile His Gly Leu 610 615 620 Tyr Arg Ser Ser Asp Lys
Thr Gly Gly Tyr Trp Lys Ile Thr Met Asn 625 630 635 640 Asp Gly Ser
Thr Tyr Gln Ser Asp Leu Ser Lys Lys Phe Glu Tyr Asn 645 650 655 Thr
Glu Lys Pro Pro Ile Asn Ile Asp Glu Ile Lys Thr Ile Glu Ala 660 665
670 Glu Ile Asn 675 35675PRTArtificialsynthetic 35Met Glu Lys Ser
Glu Glu Ile Asn Glu Lys Asp Leu Arg Lys Lys Ser 1 5 10 15 Glu Leu
Gln Gly Thr Ala Leu Gly Asn Leu Lys Gln Ile Tyr Tyr Tyr 20 25 30
Asn Glu Lys Ala Lys Thr Glu Asn Lys Glu Ser His Asp Gln Phe Arg 35
40 45 Gln His Thr Ile Leu Phe Lys Gly Phe Phe Thr Asp His Ser Trp
Tyr 50 55 60 Asn Asp Leu Leu Val Arg Phe Asp Ser Lys Asp Ile Val
Asp Lys Tyr 65 70 75 80 Lys Gly Lys Lys Val Asp Leu Tyr Gly Ala Tyr
Ala Gly Tyr Gln Cys 85 90 95 Ala Gly Gly Thr Pro Asn Lys Thr Ala
Cys Met Tyr Gly Gly Val Thr 100 105 110 Leu His Asp Asn Asn Arg Leu
Thr Glu Glu Lys Lys Val Pro Ile Asn 115 120 125 Leu Trp Leu Asp Gly
Lys Gln Asn Thr Val Pro Leu Glu Thr Val Lys 130 135 140 Thr Asn Lys
Lys Asn Val Thr Val Gln Glu Leu Asp Leu Gln Ala Arg 145 150 155 160
Arg Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr Asn Ser Asp Val Phe Asp 165
170 175 Gly Lys Val Gln Arg Gly Leu Ile Val Phe His Thr Ser Thr Glu
Pro 180 185 190 Ser Val Asn Tyr Asp Leu Phe Gly Ala Gln Gly Gln Tyr
Ser Asn Thr 195 200 205 Leu Leu Arg Ile Tyr Arg Asp Asn Lys Thr Ile
Asn Ser Glu Asn Met 210 215 220 His Ile Asp Ile Tyr Leu Tyr Thr Ser
Gly Gly Gly Gly Ser Ser Thr 225 230 235 240 Asn Asp Asn Ile Lys Asp
Leu Leu Asp Trp Tyr Ser Ser Gly Ser Asp 245 250 255 Thr Phe Thr Asn
Ser Glu Val Leu Ala Asn Ser Arg Gly Ser Met Arg 260 265 270 Ile Lys
Asn Thr Asp Gly Ser Ile Ser Leu Ile Ala Phe Pro Ser Pro 275 280 285
Tyr Tyr Ser Pro Ala Phe Thr Lys Gly Glu Lys Val Asp Leu Asn Thr 290
295 300 Lys Arg Thr Lys Lys Ser Gln His Thr Ser Glu Gly Thr Tyr Ile
His 305 310 315 320 Phe Gln Ile Ser Gly Val Thr Asn Thr Glu Lys Leu
Pro Thr Pro Ile 325 330 335 Glu Leu Pro Leu Lys Val Lys Val His Gly
Lys Asp Ser Pro Leu Lys 340 345 350 Tyr Trp Pro Lys Phe Asp Lys Lys
Gln Leu Ala Ile Ser Thr Leu Asp 355 360 365 Phe Glu Ile Arg His Gln
Leu Thr Gln Ile His Gly Leu Tyr Arg Ser 370 375 380 Ser Asp Lys Thr
Gly Gly Tyr Trp Lys Ile Thr Met Asn Asp Gly Ser 385 390 395 400 Thr
Tyr Gln Ser Asp Leu Ser Lys Lys Phe Glu Tyr Asn Thr Glu Lys 405 410
415 Pro Pro Ile Asn Ile Asp Glu Ile Lys Thr Ile Glu Ala Glu Ile Asn
420 425 430 Gly Gly Gly Ser Glu Ser Gln Pro Asp Pro Lys Pro Asp Glu
Leu His 435 440 445 Lys Ser Ser Lys Phe Thr Gly Leu Met Glu Asn Met
Lys Val Leu Tyr 450 455 460 Asp Asp Asn His Val Ser Ala Ile Asn Val
Lys Ser Ile Asp Gln Phe 465 470 475 480 Arg Tyr Phe Asp Leu Ile Tyr
Ser Ile Lys Asp Thr Lys Leu Gly Asn 485 490 495 Tyr Asp Asn Val Arg
Val Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys 500 505 510 Tyr Lys Asp
Lys Tyr Val Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln 515 520 525 Cys
Ala Phe Ser Lys Lys Thr Asn Asp Ile Asn Ser His Gln Thr Asp 530 535
540 Lys Arg Lys Thr Cys Met Tyr Gly Gly Val Thr Glu His Asn Gly Asn
545 550 555 560 Gln Leu Asp Lys Tyr Arg Ser Ile Thr Val Arg Val Phe
Glu Asp Gly 565 570 575 Lys Asn Leu Leu Ser Phe Asp Val Gln Thr Asn
Lys Lys Lys Val Thr 580 585 590 Ala Gln Glu Leu Asp Tyr Leu Thr Arg
His Tyr Leu Val Lys Asn Lys 595 600 605 Lys Leu Tyr Glu Phe Asn Asn
Ser Pro Tyr Glu Thr Gly Tyr Ile Lys 610 615 620 Phe Ile Glu Asn Glu
Asn Ser Phe Trp Tyr Asp Met Met Pro Ala Pro 625 630 635 640 Gly Asp
Lys Phe Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn 645 650 655
Lys Met Val Asp Ser Lys Asp Val Lys Ile Glu Val Tyr Leu Thr Thr 660
665 670 Lys Lys Lys 675 36675PRTArtificialsynthetic 36Met Ser Thr
Asn Asp Asn Ile Lys Asp Leu Leu Asp Trp Tyr Ser Ser 1 5 10 15 Gly
Ser Asp Thr Phe Thr Asn Ser Glu Val Leu Ala Asn Ser Arg Gly 20 25
30 Ser Met Arg Ile Lys Asn Thr Asp Gly Ser Ile Ser Leu Ile Ala Phe
35 40 45 Pro Ser Pro Tyr Tyr Ser Pro Ala Phe Thr Lys Gly Glu Lys
Val Asp 50 55 60 Leu Asn Thr Lys Arg Thr Lys Lys Ser Gln His Thr
Ser Glu Gly Thr 65 70 75 80 Tyr Ile His Phe Gln Ile Ser Gly Val Thr
Asn Thr Glu Lys Leu Pro 85 90 95 Thr Pro Ile Glu Leu Pro Leu Lys
Val Lys Val His Gly Lys Asp Ser 100 105 110 Pro Leu Lys Tyr Trp Pro
Lys Phe Asp Lys Lys Gln Leu Ala Ile Ser 115 120 125 Thr Leu Asp Phe
Glu Ile Arg His Gln Leu Thr Gln Ile His Gly Leu 130 135 140 Tyr Arg
Ser Ser Asp Lys Thr Gly Gly Tyr Trp Lys Ile Thr Met Asn 145 150 155
160 Asp Gly Ser Thr Tyr Gln Ser Asp Leu Ser Lys Lys Phe Glu Tyr Asn
165 170 175 Thr Glu Lys Pro Pro Ile Asn Ile Asp Glu Ile Lys Thr Ile
Glu Ala 180 185 190 Glu Ile Asn Gly Gly Gly Ser Glu Lys Ser Glu Glu
Ile Asn Glu Lys 195 200 205 Asp Leu Arg Lys Lys Ser Glu Leu Gln Gly
Thr Ala Leu Gly Asn Leu 210 215 220 Lys Gln Ile Tyr Tyr Tyr Asn Glu
Lys Ala Lys Thr Glu Asn Lys Glu 225 230 235 240 Ser His Asp Gln Phe
Arg Gln His Thr Ile Leu Phe Lys Gly Phe Phe 245 250 255 Thr Asp His
Ser Trp Tyr Asn Asp Leu Leu Val Arg Phe Asp Ser Lys 260 265 270 Asp
Ile Val Asp Lys Tyr Lys Gly Lys Lys Val Asp Leu Tyr Gly Ala 275 280
285 Tyr Ala Gly Tyr Gln Cys Ala Gly Gly Thr Pro Asn Lys Thr Ala Cys
290 295 300 Met Tyr Gly Gly Val Thr Leu His Asp Asn Asn Arg Leu Thr
Glu Glu 305 310 315 320 Lys Lys Val Pro Ile Asn Leu Trp Leu Asp Gly
Lys Gln Asn Thr Val 325 330 335 Pro Leu Glu Thr Val Lys Thr Asn Lys
Lys Asn Val Thr Val Gln Glu 340 345 350 Leu Asp Leu Gln Ala Arg Arg
Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr 355 360 365 Asn Ser Asp Val Phe
Asp Gly Lys Val Gln Arg Gly Leu Ile Val Phe 370 375 380 His Thr Ser
Thr Glu Pro Ser Val Asn Tyr Asp Leu Phe Gly Ala Gln 385 390 395 400
Gly Gln Tyr Ser Asn Thr Leu Leu Arg Ile Tyr Arg Asp Asn Lys Thr 405
410 415 Ile Asn Ser Glu Asn Met His Ile Asp Ile Tyr Leu Tyr Thr Ser
Gly 420 425 430 Gly Gly Gly Ser Glu Ser Gln Pro Asp Pro Lys Pro Asp
Glu Leu His 435 440 445 Lys Ser Ser Lys Phe Thr Gly Leu Met Glu Asn
Met Lys Val Leu Tyr 450 455 460 Asp Asp Asn His Val Ser Ala Ile Asn
Val Lys Ser Ile Asp Gln Phe 465 470 475 480 Arg Tyr Phe Asp Leu Ile
Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn 485 490 495 Tyr Asp Asn Val
Arg Val Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys 500 505 510 Tyr Lys
Asp Lys Tyr Val Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln 515 520 525
Cys Ala Phe Ser Lys Lys Thr Asn Asp Ile Asn Ser His Gln Thr Asp 530
535 540 Lys Arg Lys Thr Cys Met Tyr Gly Gly Val Thr Glu His Asn Gly
Asn 545 550 555 560 Gln Leu Asp Lys Tyr Arg Ser Ile Thr Val Arg Val
Phe Glu Asp Gly 565 570 575 Lys Asn Leu Leu Ser Phe Asp Val Gln Thr
Asn Lys Lys Lys Val Thr 580 585 590 Ala Gln Glu Leu Asp Tyr Leu Thr
Arg His Tyr Leu Val Lys Asn Lys 595 600 605 Lys Leu Tyr Glu Phe Asn
Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys 610 615 620 Phe Ile Glu Asn
Glu Asn Ser Phe Trp Tyr Asp Met Met Pro Ala Pro 625 630 635 640 Gly
Asp Lys Phe Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn 645 650
655 Lys Met Val Asp Ser Lys Asp Val Lys Ile Glu Val Tyr Leu Thr Thr
660 665 670 Lys Lys Lys 675 37675PRTArtificialsynthetic 37Met Ser
Thr Asn Asp Asn Ile Lys Asp Leu Leu Asp Trp Tyr Ser Ser 1 5 10 15
Gly Ser Asp Thr Phe Thr Asn Ser Glu Val Leu Ala Asn Ser Arg Gly 20
25 30 Ser Met Arg Ile Lys Asn Thr Asp Gly Ser Ile Ser Leu Ile Ala
Phe 35 40 45 Pro Ser Pro Tyr Tyr Ser Pro Ala Phe Thr Lys Gly Glu
Lys Val Asp 50 55 60 Leu Asn Thr Lys Arg Thr Lys Lys Ser Gln His
Thr Ser Glu Gly Thr 65 70 75 80 Tyr Ile His Phe Gln Ile Ser Gly Val
Thr Asn Thr Glu Lys Leu Pro 85 90 95 Thr Pro Ile Glu Leu Pro Leu
Lys Val Lys Val His Gly Lys Asp Ser 100 105 110 Pro Leu Lys Tyr Trp
Pro Lys Phe Asp Lys Lys Gln Leu Ala Ile Ser 115 120 125 Thr Leu Asp
Phe Glu Ile Arg His Gln Leu Thr Gln Ile His Gly Leu 130 135 140 Tyr
Arg Ser Ser Asp Lys Thr Gly Gly Tyr Trp Lys Ile Thr Met Asn 145 150
155 160 Asp Gly Ser Thr Tyr Gln Ser Asp Leu Ser Lys Lys Phe Glu Tyr
Asn 165 170 175 Thr Glu Lys Pro Pro Ile Asn Ile Asp Glu Ile Lys Thr
Ile Glu Ala 180 185 190 Glu Ile Asn Gly Gly Gly Gly Ser Glu Ser Gln
Pro Asp Pro Lys Pro 195 200 205 Asp Glu Leu His Lys Ser Ser Lys Phe
Thr Gly Leu Met Glu Asn Met 210 215 220 Lys Val Leu Tyr Asp Asp Asn
His Val Ser Ala Ile Asn Val Lys Ser 225 230 235 240 Ile Asp Gln Phe
Arg Tyr Phe Asp Leu Ile Tyr Ser Ile Lys Asp Thr 245 250 255 Lys Leu
Gly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys Asn Lys Asp 260 265 270
Leu Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe Gly Ala Asn 275
280 285 Ala Tyr Tyr Gln Cys Ala Phe Ser Lys Lys Thr Asn Asp Ile Asn
Ser 290 295 300 His Gln Thr Asp Lys Arg Lys Thr Cys Met Tyr Gly Gly
Val Thr Glu 305 310 315 320 His Asn Gly Asn Gln Leu Asp Lys Tyr Arg
Ser Ile Thr Val Arg Val 325 330 335 Phe Glu Asp Gly Lys Asn Leu Leu
Ser Phe Asp Val Gln Thr Asn Lys 340 345 350 Lys Lys Val Thr Ala Gln
Glu Leu Asp Tyr Leu Thr Arg His Tyr Leu 355 360 365 Val Lys Asn Lys
Lys Leu Tyr Glu Phe Asn Asn Ser Pro Tyr Glu Thr 370 375 380 Gly Tyr
Ile Lys Phe Ile Glu Asn Glu Asn Ser Phe Trp Tyr Asp Met 385 390 395
400 Met Pro Ala Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr Leu Met Met
405 410 415 Tyr Asn Asp Asn Lys Met Val Asp Ser Lys Asp Val Lys Ile
Glu Val 420 425 430 Tyr Leu Thr Thr Lys Lys Lys Gly Gly Gly Ser Glu
Lys Ser Glu Glu 435 440 445 Ile Asn Glu Lys Asp Leu Arg Lys Lys Ser
Glu Leu Gln Gly Thr Ala 450 455 460 Leu Gly Asn Leu Lys Gln Ile Tyr
Tyr Tyr Asn Glu Lys Ala Lys Thr 465 470 475 480 Glu Asn Lys Glu Ser
His Asp Gln Phe Arg Gln His Thr Ile Leu Phe 485 490 495 Lys Gly Phe
Phe Thr Asp His Ser Trp Tyr Asn Asp Leu Leu Val Arg 500 505 510 Phe
Asp Ser Lys Asp Ile Val Asp Lys Tyr Lys Gly Lys Lys Val Asp 515 520
525 Leu Tyr Gly Ala Tyr Ala Gly Tyr Gln Cys Ala Gly Gly Thr Pro Asn
530 535 540 Lys Thr Ala Cys Met Tyr Gly Gly Val Thr Leu His Asp Asn
Asn Arg 545 550 555 560 Leu Thr Glu Glu Lys Lys Val Pro Ile Asn Leu
Trp
Leu Asp Gly Lys 565 570 575 Gln Asn Thr Val Pro Leu Glu Thr Val Lys
Thr Asn Lys Lys Asn Val 580 585 590 Thr Val Gln Glu Leu Asp Leu Gln
Ala Arg Arg Tyr Leu Gln Glu Lys 595 600 605 Tyr Asn Leu Tyr Asn Ser
Asp Val Phe Asp Gly Lys Val Gln Arg Gly 610 615 620 Leu Ile Val Phe
His Thr Ser Thr Glu Pro Ser Val Asn Tyr Asp Leu 625 630 635 640 Phe
Gly Ala Gln Gly Gln Tyr Ser Asn Thr Leu Leu Arg Ile Tyr Arg 645 650
655 Asp Asn Lys Thr Ile Asn Ser Glu Asn Met His Ile Asp Ile Tyr Leu
660 665 670 Tyr Thr Ser 675 3821PRTStaphylococcus aureus 38Met Gly
Ile Ile Ala Gly Ile Ile Lys Val Ile Lys Ser Leu Ile Glu 1 5 10 15
Gln Phe Thr Gly Lys 20 3926PRTArtificialsyntheticVARIANT(5)..(5)Xaa
is isoleucine, glycine, or alanineVARIANT(6)..(6)Xaa is isoleucine,
glycine, or alanineVARIANT(9)..(9)Xaa is isoleucine, glycine, or
alanineVARIANT(12)..(12)Xaa is leucine, glycine, or
valineVARIANT(13)..(13)Xaa is valine, glycine, or
alanineVARIANT(16)..(16)Xaa is isoleucine, glycine, or
alanineVARIANT(17)..(17)Xaa is isoleucine, glycine, or
alanineVARIANT(20)..(20)Xaa is valine, glycine, or alanine 39Met
Ala Gln Asp Xaa Xaa Ser Thr Xaa Gly Asp Xaa Xaa Lys Trp Xaa 1 5 10
15 Xaa Asp Thr Xaa Asn Lys Phe Thr Lys Lys 20 25
4021PRTArtificialsyntheticVARIANT(1)..(1)Xaa is methionine,
glycine, or alanineVARIANT(3)..(3)Xaa is isoleucine, glycine, or
alanineVARIANT(4)..(4)Xaa is isoleucine, glycine, or
alanineVARIANT(7)..(7)Xaa is isoleucine, glycine, or
alanineVARIANT(8)..(8)Xaa is isoleucine, glycine, or
alanineVARIANT(10)..(10)Xaa is valine, glycine, or
alanineVARIANT(11)..(11)Xaa is isoleucine, glycine, or
alanineVARIANT(14)..(14)Xaa is leucine, glycine, or
alanineVARIANT(15)..(15)Xaa is isoleucine, glycine, or
alanineVARIANT(18)..(18)Xaa is phenylalanine, glycine, or alanine
40Xaa Gly Xaa Xaa Ala Gly Xaa Xaa Lys Xaa Xaa Lys Ser Xaa Xaa Glu 1
5 10 15 Gln Xaa Thr Gly Lys 20
4121PRTArtificialsyntheticVARIANT(1)..(1)Xaa is methionine,
glycine, or alanineVARIANT(3)..(3)Xaa is isoleucine, glycine, or
alanineVARIANT(4)..(4)Xaa is isoleucine, glycine, or
alanineVARIANT(7)..(7)Xaa is isoleucine, glycine, or
alanineVARIANT(8)..(8)Xaa is isoleucine, glycine, or
alanineVARIANT(10)..(10)Xaa is phenylalanine, glycine, or
alanineVARIANT(11)..(11)Xaa is isoleucine, glycine, or
alanineVARIANT(14)..(14)Xaa is leucine, glycine, or
alanineVARIANT(15)..(15)Xaa is isoleucine, glycine, or
alanineVARIANT(18)..(18)Xaa is phenylalanine, glycine, or alanine
41Xaa Gly Xaa Xaa Ala Gly Xaa Xaa Lys Xaa Xaa Lys Gly Xaa Xaa Glu 1
5 10 15 Lys Xaa Thr Gly Lys 20
4222PRTArtificialsyntheticVARIANT(1)..(1)Xaa is methionine,
glycine, or alanineVARIANT(3)..(3)Xaa is phenylalanine, glycine, or
alanineVARIANT(4)..(4)Xaa is valine, glycine, or
alanineVARIANT(7)..(7)Xaa is leucine, glycine, or
alanineVARIANT(8)..(8)Xaa is phenylalanine, glycine, or
alanineVARIANT(10)..(10)Xaa is phenylalanine, glycine, or
alanineVARIANT(11)..(11)Xaa is phenylalanine, glycine, or
alanineVARIANT(14)..(14)Xaa is leucine, glycine, or
alanineVARIANT(15)..(15)Xaa is leucine, glycine, or
alanineVARIANT(18)..(18)Xaa is phenylalanine, glycine, or
alanineVARIANT(19)..(19)Xaa is leucine, glycine, or alanine 42Xaa
Glu Xaa Xaa Ala Lys Xaa Xaa Lys Xaa Xaa Lys Asp Xaa Xaa Gly 1 5 10
15 Lys Xaa Xaa Gly Asn Asn 20
4320PRTArtificialsyntheticmisc_feature(1)..(1)Xaa is methionine,
glycine, or alaninemisc_feature(3)..(3)Xaa is isoleucine, glycine,
or alaninemisc_feature(4)..(4)Xaa is valine, glycine, or
alaninemisc_feature(7)..(7)Xaa is isoleucine, glycine, or
alaninemisc_feature(8)..(8)Xaa is isoleucine, glycine, or
alaninemisc_feature(10)..(10)Xaa is isoleucine, glycine, or
alaninemisc_feature(11)..(11)Xaa is isoleucine, glycine, or
alaninemisc_feature(14)..(14)Xaa is isoleucine, glycine, or
alaninemisc_feature(15)..(15)Xaa is isoleucine, glycine, or
alaninemisc_feature(17)..(17)Xaa is isoleucine, glycine, or
alaninemisc_feature(18)..(18)Xaa is phenylalanine, glycine, or
alanine 43Xaa Ala Xaa Xaa Gly Thr Xaa Xaa Lys Xaa Xaa Lys Ala Xaa
Xaa Asp 1 5 10 15 Xaa Xaa Ala Lys 20
4444PRTArtificialsyntheticVariant(4)..(4)Xaa is leucine, glycine,
or alanineVariant(5)..(5)Xaa is phenylalanine, glycine, or
alanineVariant(8)..(8)Xaa is isoleucine, glycine, or
alanineVariant(12)..(12)Xaa is valine, glycine, or
alanineVariant(16)..(16)Xaa is isoleucine, glycine, or alanine
44Met Glu Gly Xaa Xaa Asn Ala Xaa Lys Asp Thr Xaa Thr Ala Ala Xaa 1
5 10 15 Asn Asn Asp Gly Ala Lys Leu Gly Thr Ser Ile Val Asn Ile Val
Glu 20 25 30 Asn Gly Val Gly Leu Leu Ser Lys Leu Phe Gly Phe 35 40
4544PRTArtificialsyntheticVariant(4)..(4)Xaa is leucine, glycine,
or alanineVariant(8)..(8)Xaa is isoleucine, glycine, or
alanineVariant(12)..(12)Xaa is valine, glycine, or
alanineVariant(23)..(23)Xaa is leucine, glycine, or alanine 45Met
Thr Gly Xaa Ala Glu Ala Xaa Ala Asn Thr Xaa Gln Ala Ala Gln 1 5 10
15 Gln His Asp Ser Val Lys Xaa Gly Thr Ser Ile Val Asp Ile Val Ala
20 25 30 Asn Gly Val Gly Leu Leu Gly Lys Leu Phe Gly Phe 35 40
4662PRTArtificialsynthetic 46Ala Asp Ser Asp Ile Asn Ile Lys Thr
Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly
Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys
Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys
Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala 50 55 60
47284PRTStaphylococcus aureus 47Asp Asn Asn Ile Glu Asn Ile Gly Asp
Gly Ala Glu Val Val Lys Arg 1 5 10 15 Thr Glu Asp Thr Ser Ser Asp
Lys Trp Gly Val Thr Gln Asn Ile Gln 20 25 30 Phe Asp Phe Val Lys
Asp Lys Lys Tyr Asn Lys Asp Ala Leu Ile Leu 35 40 45 Lys Met Gln
Gly Phe Ile Asn Ser Lys Thr Thr Tyr Tyr Asn Tyr Lys 50 55 60 Asn
Thr Asp His Ile Lys Ala Met Arg Trp Pro Phe Gln Tyr Asn Ile 65 70
75 80 Gly Leu Lys Thr Asn Asp Pro Asn Val Asp Leu Ile Asn Tyr Leu
Pro 85 90 95 Lys Asn Lys Ile Asp Ser Val Asn Val Ser Gln Thr Leu
Gly Tyr Asn 100 105 110 Ile Gly Gly Asn Phe Asn Ser Gly Pro Ser Thr
Gly Gly Asn Gly Ser 115 120 125 Phe Asn Tyr Ser Lys Thr Ile Ser Tyr
Asn Gln Gln Asn Tyr Ile Ser 130 135 140 Glu Val Glu His Gln Asn Ser
Lys Ser Val Gln Trp Gly Ile Lys Ala 145 150 155 160 Asn Ser Phe Ile
Thr Ser Leu Gly Lys Met Ser Gly His Asp Pro Asn 165 170 175 Leu Phe
Val Gly Tyr Lys Pro Tyr Ser Gln Asn Pro Arg Asp Tyr Phe 180 185 190
Val Pro Asp Asn Glu Leu Pro Pro Leu Val His Ser Gly Phe Asn Pro 195
200 205 Ser Phe Ile Ala Thr Val Ser His Glu Lys Gly Ser Gly Asp Thr
Ser 210 215 220 Glu Phe Glu Ile Thr Tyr Gly Arg Asn Met Asp Val Thr
His Ala Thr 225 230 235 240 Arg Arg Thr Thr His Tyr Gly Asn Ser Tyr
Leu Glu Gly Ser Arg Ile 245 250 255 His Asn Ala Phe Val Asn Arg Asn
Tyr Thr Val Lys Tyr Glu Val Asn 260 265 270 Trp Lys Thr His Glu Ile
Lys Val Lys Gly His Asn 275 280 48301PRTStaphylococcus aureus 48Ala
Gln His Ile Thr Pro Val Ser Glu Lys Lys Val Asp Asp Lys Ile 1 5 10
15 Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Ser Asp Lys Leu Lys Ile
20 25 30 Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr
Asp Lys 35 40 45 Asp Thr Leu Ile Leu Lys Ala Ala Gly Asn Ile Tyr
Ser Gly Tyr Thr 50 55 60 Lys Pro Asn Pro Lys Asp Thr Ile Ser Ser
Gln Phe Tyr Trp Gly Ser 65 70 75 80 Lys Tyr Asn Ile Ser Ile Asn Ser
Asp Ser Asn Asp Ser Val Asn Val 85 90 95 Val Asp Tyr Ala Pro Lys
Asn Gln Asn Glu Glu Phe Gln Val Gln Gln 100 105 110 Thr Val Gly Tyr
Ser Tyr Gly Gly Asp Ile Asn Ile Ser Asn Gly Leu 115 120 125 Ser Gly
Gly Gly Asn Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn Tyr 130 135 140
Lys Gln Glu Ser Tyr Arg Thr Ser Leu Asp Lys Arg Thr Asn Phe Lys 145
150 155 160 Lys Ile Gly Trp Asp Val Glu Ala His Lys Ile Met Asn Asn
Gly Trp 165 170 175 Gly Pro Tyr Gly Arg Asp Ser Tyr His Ser Thr Tyr
Gly Asn Glu Met 180 185 190 Phe Leu Gly Ser Arg Gln Ser Asn Leu Asn
Ala Gly Gln Asn Phe Leu 195 200 205 Glu Tyr His Lys Met Pro Val Leu
Ser Arg Gly Asn Phe Asn Pro Glu 210 215 220 Phe Ile Gly Val Leu Ser
Arg Lys Gln Asn Ala Ala Lys Lys Ser Lys 225 230 235 240 Ile Thr Val
Thr Tyr Gln Arg Glu Met Asp Arg Tyr Thr Asn Phe Trp 245 250 255 Asn
Gln Leu His Trp Ile Gly Asn Asn Tyr Lys Asp Glu Asn Arg Ala 260 265
270 Thr His Thr Ser Ile Tyr Glu Val Asp Trp Glu Asn His Thr Val Lys
275 280 285 Leu Ile Asp Thr Gln Ser Lys Glu Lys Asn Pro Met Ser 290
295 300 49240PRTArtificialsynthetic 49Met Glu Ser Gln Pro Asp Pro
Lys Pro Asp Glu Leu His Lys Ser Ser 1 5 10 15 Lys Phe Thr Gly Leu
Met Glu Asn Met Lys Val Leu Tyr Asp Asp Asn 20 25 30 His Val Ser
Ala Ile Asn Val Lys Ser Ile Asp Gln Phe Arg Tyr Phe 35 40 45 Asp
Leu Ile Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn 50 55
60 Val Arg Val Glu Phe Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp
65 70 75 80 Lys Tyr Val Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln Cys
Ala Phe 85 90 95 Ser Lys Lys Thr Asn Asp Ile Asn Ser His Gln Thr
Asp Lys Arg Lys 100 105 110 Thr Cys Met Tyr Gly Gly Val Thr Glu His
Asn Gly Asn Gln Leu Asp 115 120 125 Lys Tyr Arg Ser Ile Thr Val Arg
Val Phe Glu Asp Gly Lys Asn Leu 130 135 140 Leu Ser Phe Asp Val Gln
Thr Asn Lys Lys Lys Val Thr Ala Gln Glu 145 150 155 160 Leu Asp Tyr
Leu Thr Arg His Tyr Leu Val Lys Asn Lys Lys Leu Tyr 165 170 175 Glu
Phe Asn Asn Ser Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile Glu 180 185
190 Asn Glu Asn Ser Phe Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys
195 200 205 Phe Asp Gln Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys
Met Val 210 215 220 Asp Ser Lys Asp Val Lys Ile Glu Val Tyr Leu Thr
Thr Lys Lys Lys 225 230 235 240 50232PRTArtificialsynthetic 50Glu
Lys Ser Glu Glu Ile Asn Glu Lys Asp Leu Arg Lys Lys Ser Glu 1 5 10
15 Leu Gln Gly Thr Ala Leu Gly Asn Leu Lys Gln Ile Tyr Tyr Tyr Asn
20 25 30 Glu Lys Ala Lys Thr Glu Asn Lys Glu Ser His Asp Gln Phe
Arg Gln 35 40 45 His Thr Ile Leu Phe Lys Gly Phe Phe Thr Asp His
Ser Trp Tyr Asn 50 55 60 Asp Leu Leu Val Arg Phe Asp Ser Lys Asp
Ile Val Asp Lys Tyr Lys 65 70 75 80 Gly Lys Lys Val Asp Leu Tyr Gly
Ala Tyr Ala Gly Tyr Gln Cys Ala 85 90 95 Gly Gly Thr Pro Asn Lys
Thr Ala Cys Met Tyr Gly Gly Val Thr Leu 100 105 110 His Asp Asn Asn
Arg Leu Thr Glu Glu Lys Lys Val Pro Ile Asn Leu 115 120 125 Trp Leu
Asp Gly Lys Gln Asn Thr Val Pro Leu Glu Thr Val Lys Thr 130 135 140
Asn Lys Lys Asn Val Thr Val Gln Glu Leu Asp Leu Gln Ala Arg Arg 145
150 155 160 Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr Asn Ser Asp Val Phe
Asp Gly 165 170 175 Lys Val Gln Arg Gly Leu Ile Val Phe His Thr Ser
Thr Glu Pro Ser 180 185 190 Val Asn Tyr Asp Leu Phe Gly Ala Gln Gly
Gln Tyr Ser Asn Thr Leu 195 200 205 Leu Arg Ile Tyr Arg Asp Asn Lys
Thr Ile Asn Ser Glu Asn Met His 210 215 220 Ile Asp Ile Tyr Leu Tyr
Thr Ser 225 230 51195PRTArtificialsynthetic 51Met Ser Thr Asn Asp
Asn Ile Lys Asp Leu Leu Asp Trp Tyr Ser Ser 1 5 10 15 Gly Ser Asp
Thr Phe Thr Asn Ser Glu Val Leu Ala Asn Ser Arg Gly 20 25 30 Ser
Met Arg Ile Lys Asn Thr Asp Gly Ser Ile Ser Leu Ile Ala Phe 35 40
45 Pro Ser Pro Tyr Tyr Ser Pro Ala Phe Thr Lys Gly Glu Lys Val Asp
50 55 60 Leu Asn Thr Lys Arg Thr Lys Lys Ser Gln His Thr Ser Glu
Gly Thr 65 70 75 80 Tyr Ile His Phe Gln Ile Ser Gly Val Thr Asn Thr
Glu Lys Leu Pro 85 90 95 Thr Pro Ile Glu Leu Pro Leu Lys Val Lys
Val His Gly Lys Asp Ser 100 105 110 Pro Leu Lys Tyr Trp Pro Lys Phe
Asp Lys Lys Gln Leu Ala Ile Ser 115 120 125 Thr Leu Asp Phe Glu Ile
Arg His Gln Leu Thr Gln Ile His Gly Leu 130 135 140 Tyr Arg Ser Ser
Asp Lys Thr Gly Gly Tyr Trp Lys Ile Thr Met Asn 145 150 155 160 Asp
Gly Ser Thr Tyr Gln Ser Asp Leu Ser Lys Lys Phe Glu Tyr Asn 165 170
175 Thr Glu Lys Pro Pro Ile Asn Ile Asp Glu Ile Lys Thr Ile Glu Ala
180 185 190 Glu Ile Asn 195 5222PRTStaphylococcus aureus 52Met Asp
Phe Thr Gly Val Ile Thr Ser Ile Ile Asp Leu Ile Lys Thr 1 5 10 15
Cys Ile Gln Ala Phe Gly 20
5322PRTArtificialsyntheticVariant(3)..(3)Xaa is phenylalanine,
glycine, or alanineVariant(6)..(6)Xaa is valine, glycine, or
alanineVariant(7)..(7)Xaa is isoleucine, glycine, or
alanineVariant(10)..(10)Xaa is isoleucine, glycine, or
alanineVariant(11)..(11)Xaa is isoleucine, glycine, or
alanineVariant(13)..(13)Xaa is leucine, glycine, or
alanineVariant(14)..(14)Xaa is isoleucine, glycine, or
alanineVariant(17)..(17)Xaa is cysteine, glycine, or
alanineVariant(18)..(18)Xaa is isoleucine, glycine, or alanine
53Met Asp Xaa Thr Gly Xaa Xaa Thr Ser Xaa Xaa Asp Xaa Xaa Lys Thr 1
5 10 15 Xaa Xaa Gln Ala Phe Gly 20 54284PRTArtificialsynthetic
54Asp Asn Asn Ile Glu Asn Ile Gly Asp Gly Ala Glu Val Val Lys Arg 1
5 10 15 Thr Glu Asp Thr Ser Ser Asp Lys Trp Gly Val Phe Gln Asn Ile
Gln 20 25 30 Phe Asp Phe Val Lys Asp Lys Lys Tyr Asn Lys Asp Ala
Leu Ile Leu 35 40 45 Lys Met Gln Gly Phe Ile Asn Ser Lys Thr Thr
Tyr Tyr Asn Tyr Lys 50 55 60 Asn Thr Asp His Ile Lys Ala Met Arg
Trp Pro Phe Gln Tyr Asn Ile 65 70 75 80 Gly Leu Lys Thr Asn Asp Pro
Asn Val Asp Leu Ile Asn Tyr Leu Pro 85 90 95 Ala Asn Lys Ile Asp
Ser Val Asn Val Ser Gln Thr Leu Gly Tyr Asn 100 105 110 Ile Gly Gly
Asn Phe Asn Ser Gly Pro Ser Thr Gly Gly Asn Gly Ser 115 120 125 Phe
Asn Tyr Ser Lys Thr Ile Ser Tyr Asn Gln Gln Asn Tyr Ile Ser 130 135
140 Glu Val Glu His Gln Asn Ser Lys Ser Val Gln Trp Gly Ile Lys Ala
145 150 155 160 Asn Ser Phe Ile Thr Ser Leu Gly Lys Met Ser Gly His
Asp Pro Asn 165 170 175 Leu Phe Val Gly Tyr Lys Pro Tyr Ser Gln Asn
Pro Arg Asp Tyr Phe 180 185 190 Val Pro Asp Asn Glu Leu Pro Pro Leu
Val His Ser Gly Phe Asn Pro 195 200 205 Ala Phe Ile Ala Thr Val Ser
His Glu Lys Gly Ser Gly Asp Thr Ser 210 215 220 Glu Phe Glu Ile Thr
Tyr Gly Arg Asn Met Asp Val Thr His Ala Thr 225 230 235 240 Arg Arg
Thr Thr His Tyr Gly Asn Ser Tyr Leu Glu Gly Ser Arg Ile 245 250 255
His Asn Ala Phe Val Asn Arg Asn Tyr Thr Val Lys Tyr Glu Val Asn 260
265 270 Trp Lys Thr His Glu Ile Lys Val Lys Gly His Asn 275 280
55301PRTArtificialsynthetic 55Ala Gln His Ile Thr Pro Val Ser Glu
Lys Lys Val Asp Asp Lys Ile 1 5 10 15 Thr Leu Tyr Lys Thr Thr Ala
Thr Ser Asp Ser Asp Lys Leu Lys Ile 20 25 30 Ser Gln Ile Leu Thr
Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp Lys 35 40 45 Asp Thr Leu
Ile Leu Lys Ala Ala Gly Asn Ile Tyr Ser Gly Tyr Thr 50 55 60 Lys
Pro Asn Pro Lys Asp Thr Ile Ser Ser Gln Phe Tyr Trp Gly Ser 65 70
75 80 Lys Tyr Asn Ile Ser Ile Asn Ser Asp Ser Asn Asp Ser Val Asn
Val 85 90 95 Val Asp Tyr Ala Pro Ala Asn Gln Asn Glu Glu Phe Gln
Val Gln Gln 100 105 110 Thr Val Gly Tyr Ser Tyr Gly Gly Asp Ile Asn
Ile Ser Asn Gly Leu 115 120 125 Ser Gly Gly Gly Asn Gly Ser Lys Ser
Phe Ser Glu Thr Ile Asn Tyr 130 135 140 Lys Gln Glu Ser Tyr Arg Thr
Ser Leu Asp Lys Arg Thr Asn Phe Lys 145 150 155 160 Lys Ile Gly Trp
Asp Val Glu Ala His Lys Ile Met Asn Asn Gly Trp 165 170 175 Gly Pro
Tyr Gly Arg Asp Ser Tyr His Ser Thr Tyr Gly Asn Glu Met 180 185 190
Phe Leu Gly Ser Arg Gln Ser Asn Leu Asn Ala Gly Gln Asn Phe Leu 195
200 205 Glu Tyr His Lys Met Pro Val Leu Ser Arg Gly Asn Phe Asn Pro
Glu 210 215 220 Phe Ile Gly Val Leu Ser Arg Lys Gln Asn Ala Ala Lys
Lys Ser Lys 225 230 235 240 Ile Thr Val Thr Tyr Gln Arg Glu Met Asp
Arg Tyr Thr Asn Phe Trp 245 250 255 Asn Gln Leu His Trp Ile Gly Asn
Asn Tyr Lys Asp Glu Asn Arg Ala 260 265 270 Thr His Thr Ser Ile Tyr
Glu Val Asp Trp Glu Asn His Thr Val Lys 275 280 285 Leu Ile Asp Thr
Gln Ser Lys Glu Lys Asn Pro Met Ser 290 295 300 564PRTArtificial
SequenceSynthetic linker 56Gly Gly Gly Ser 1 575PRTArtificial
SequenceSynthetic linker 57Gly Gly Gly Gly Ser 1 5
5810PRTStaphylococcus aureus 58Val Ile Leu Ile Phe Ile Ile Ile Ile
Met 1 5 10 5910PRTStaphylococcus aureus 59Phe Ile Leu Ile Phe Ile
Ile Ile Ile Met 1 5 10 6010PRTStaphylococcus aureus 60Phe Phe Leu
Leu Phe Phe Val Leu Phe Met 1 5 10 6111PRTStaphylococcus aureus
61Ile Ile Ile Ile Ile Phe Ile Val Ile Ile Met 1 5 10
626PRTStaphylococcus aureus 62Leu Ala Ile Met Val Phe 1 5
636PRTStaphylococcus aureus 63Leu Ala Ile Met Val Ala 1 5
647PRTStaphylococcus aureus 64Ile Ile Ile Val Val Ile Ile 1 5
6522PRTStaphylococcus aureus 65Met Asp Phe Thr Gly Val Ile Thr Ser
Ile Ile Asp Leu Ile Lys Thr 1 5 10 15 Cys Ile Gln Ala Phe Gly 20
669PRTStaphylococcus aureus 66Leu Val Cys Ile Phe Ile Ile Ile Ile 1
5
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